Inhibition of secretion from non-neuronal cells

ABSTRACT

The present invention relates to treatment of disease by inhibition of cellular secretory processes, to agents and compositions therefor, and to manufacture of those agents and compositions. The present invention relates particularly, to treatment of disease dependent upon the exocytotic activity of endocrine cells, exocrine cells, inflammatory cells, cells of the immune system, cells of the cardiovascular system and bone cells.

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/327,855, filed on Jan. 9, 2006, which is a continuation ofU.S. patent application Ser. No. 10/088,665, filed Aug. 14, 2002, whichis a national phase entry of PCT/GB00/03681, filed Sep. 25, 2000, whichclaims the benefit of priority of GB 9922558.3, filed Sep. 23, 1999.Each of these applications is hereby incorporated by reference in theirentirety.

The present invention relates to treatment of disease by inhibition ofcellular secretory processes, to agents and compositions therefor, andto manufacture of those agents and compositions. The present inventionrelates particularly, to treatment of diseases dependent upon theexocytotic activity of endocrine cells, exocrine cells, inflammatorycells, cells of the immune system, cells of the cardiovascular systemand bone cells.

Exocytosis is the fusion of secretory vesicles with the plasma membraneand results in the discharge of vesicle content—a process also known ascell secretion. Exocytosis can be constitutive or regulated.Constitutive exocytosis is thought to occur in every cell type whereasregulated exocytosis occurs from specialised cells.

The understanding of the mechanisms involved in exocytosis has increasedrapidly, following the proposal of the SNARE hypothesis (Rothman, 1994,Nature 372, 55-63). This hypothesis describes protein markers onvesicles, which recognise target membrane markers. These so-calledcognate SNARES (denoted v-SNARE for vesicle and t-SNARE for target)facilitate docking and fusion of vesicles with the correct membranes,thus directing discharge of the vesicular contents into the appropriatecompartment. Key to the understanding of this process has been theidentification of the proteins involved. Three SNARE protein familieshave been identified for exocytosis: SNAP-25 and SNAP-23, and syntaxinsare the t-SNARE families in the membrane; and VAMPs (vesicle-associatedmembrane protein), including synaptobrevin and cellubrevin, are thev-SNARE family on secretory vesicles. Key components of the fusionmachinery including SNARES are involved in both regulated andconstitutive exocytosis (De Camilli, 1993, Nature, 364, 387-388).

The clostridial neurotoxins are proteins with molecular masses of theorder of 150 kDa. They are produced by various species of the genusClostridium, most importantly C. tetani and several strains of C.botulinum. There are at present eight different classes of theneurotoxins known: tetanus toxin and botulinum neurotoxin in itsserotypes A, B, C₁, D, E, F and G, and they all share similar structuresand modes of action. The clostridial neurotoxins are synthesized by thebacterium as a single polypeptide that is modified post-translationallyto form two polypeptide chains joined together by a disulphide bond. Thetwo chains are termed the heavy chain (H) which has a molecular mass ofapproximately 100 kDa and the light chain (LC) which has a molecularmass of approximately 50 kDa. The clostridial neurotoxins are highlyselective for neuronal cells, and bind with high affinity thereto [seeBlack, J. D. and Dolly, J. O. (1987) Selective location of acceptors forBoNT/A in the central and peripheral nervous systems. Neuroscience, 23,pp. 767-779; Habermann, E. and Dreyer, F. (1986) Clostridialneurotoxins:handling and action at the cellular and molecular level.Curr. Top. Microbiol. Immunol. 129, pp. 93-179; and Sugiyama, H. (1980)Clostridium botulinum neurotoxin. Microbiol. Rev., 44, pp. 419-448 (andinternally cited references)].

The functional requirements of neurointoxication by the clostridialneurotoxins can be assigned to specific domains within the neurotoxinstructure. The clostridial neurotoxins bind to an acceptor site on thecell membrane of the motor neuron at the neuromuscular junction and,following binding to the highly specific receptor, are internalised byan endocytotic mechanism. The specific neuromuscular junction bindingactivity of clostridial neurotoxins is known to reside in thecarboxy-terminal portion of the heavy chain component of the dichainneurotoxin molecule, a region known as H_(C). The internalisedclostridial neurotoxins possess a highly specific zinc-dependentendopeptidase activity that hydrolyses a specific peptide bond in atleast one of three protein families, synaptobrevin, syntaxin or SNAP-25,which are crucial components of the neurosecretory machinery. Thezinc-dependent endopeptidase activity of clostridial neurotoxins isfound to reside in the L-chain (LC). The amino-terminal portion of theheavy chain component of the dichain neurotoxin molecule, a region knownas H_(N), is responsible for translocation of the neurotoxin, or aportion of it containing the endopeptidase activity, across theendosomal membrane following internalisation, thus allowing access ofthe endopeptidase to the neuronal cytosol and its substrate protein(s).The result of neurointoxication is inhibition of neurotransmitterrelease from the target neuron due to prevention of release of synapticvesicle contents.

The mechanism by which the H_(N) domain effects translocation of theendopeptidase into the neuronal cytosol is not fully characterised butis believed to involve a conformational change, insertion into theendosomal membrane and formation of some form of channel or pore throughwhich the endopeptidase can gain access to the neuronal cytosol.Following binding to its specific receptor at the neuronal surfacepharmacological and morphologic evidence indicate that the clostridialneurotoxins enter the cell by endocytosis [Black & Dolly (1986) J. CellBiol. 103, 535-44] and then have to pass through a low pH step forneuron intoxication to occur [Simpson et al (1994) J. Pharmacol Exp.Ther., 269, 256-62]. Acidic pH does not activate the toxin directly viaa structural change, but is believed to trigger the process of LCmembrane translocation from the neuronal endosomal vesicle lumen to theneuronal cytosol [Montecucco et al (1994) FEBS Lett. 346, 92-98]. Thereis a general consensus that toxin-determined channels are related to thetranslocation process into the cytosol [Schiavo & Montecucco (1997) inBacterial Toxins (ed. K. Aktories)]. This model requires that the H_(N)domain forms a transmembrane hydrophobic pore across the acidic vesiclemembrane that allows the partially unfolded LC passage through to thecytosol. The requisite conformational change is believed to be triggeredby environmental factors in the neuronal endosomal compartment intowhich the neurotoxin is internalised, and a necessary feature of thebinding domain of the H_(C) is to target binding sites which enableinternalisation into the appropriate endosomal compartment. Thereforeclostridial neurotoxins have evolved to target cell surface moietiesthat fulfil this requirement.

Hormones are chemical messengers that are secreted by the endocrineglands of the body. They exercise specific physiological actions onother organs to which they are carried by the blood. The range ofprocesses regulated by hormones includes various aspects of homeostasis(e.g. insulin regulates the concentration of glucose in the blood),growth (e.g. growth hormone promotes growth and regulates fat,carbohydrate and protein metabolism), and maturation (e.g. sex hormonespromote sexual maturation and reproduction). Endocrine hyperfunctionresults in disease conditions which are caused by excessive amounts of ahormone or hormones in the bloodstream. The causes of hyperfunction areclassified as neoplastic, autoimmune, iatrogenic and inflammatory. Theendocrine hyperfunction disorders are a complex group of diseases, notonly because there is a large number of glands that can cause apathology (e.g. anterior pituitary, posterior pituitary, thyroid,parathyroid, adrenal cortex, adrenal medulla, pancreas, ovaries, testis)but because many of the glands produce more than one hormone (e.g. theanterior pituitary produces corticotrophin, prolactin, luteinizinghormone, follicle stimulating hormone, thyroid stimulating hormone andgonadotrophins). The majority of disorders that cause hormone excess aredue to neoplastic growth of hormone producing cells. However, certaintumours of non-endocrine origin can synthesise hormones causingendocrine hyperfunction disease symptoms. The hormone production underthese conditions is termed “ectopic”. Surgical removal or radiationinduced destruction of part or all of the hypersecreting tissue isfrequently the treatment of choice. However, these approaches are notalways applicable, result in complete loss of hormone production or haveto be repeated due to re-growth of the secreting tissue.

A further level of complexity in endocrine hyperfunction disordersarises in a group of conditions termed multiple endocrine neoplasia(MEN) where two or more endocrine glands are involved. The multipleendocrine neoplasia syndromes (MEN1 and MEN2) are familial conditionswith an autosomal dominant pattern of inheritance. MEN1 is characterisedby the association of parathyroid hyperplasia, pancreatic endocrinetumours, and pituitary adenomas, and has a prevalence of about 1 in10000. MEN2 is the association of medullary cell carcinoma of thethyroid and phaeochromocytoma, though parathyroid hyperplasia may alsooccur in some sufferers.

Most of the morbidity associated with MEN1 is due to the effects ofpancreatic endocrine tumours. Often surgery is not possible and thetherapeutic aim is to reduce hormone excess. Aside from reducing tumourbulk, which is often precluded, inhibition of hormone secretion is thepreferred course of action. Current procedures include subcutaneousapplication of the somatostatin analogue, octreotide. However, thisapproach is only temporarily effective, and the success diminishes overa period of months.

Many further disease states are known that involve secretion from othernon-endocrine, non-neuronal cells. It would accordingly be desirable totreat, reduce or prevent secretion by non-neuronal cells, such ashyperfunction of the endocrine cells that causes or leads to thesedisease conditions.

The activity of the botulinum neurotoxins is exclusively restricted toinhibition of neurotransmitter release from neurons. This is due to theexclusive expression of high affinity binding sites for clostridialneurotoxins on neuronal cells [see Daniels-Holgate, P. U. and Dolly, J.O. (1996) Productive and non-productive binding of botulinum neurotoxinto motor nerve endings are distinguished by its heavy chain. J.Neurosci. Res. 44, 263-271].

Non-neuronal cells do not possess the high affinity binding sites forclostridial neurotoxins, and are therefore refractory to the inhibitoryeffects of exogenously applied neurotoxin. Simple application ofclostridial neurotoxins to the surface of non-neuronal cells does nottherefore lead to inhibition of secretory vesicle exocytosis.

The productive binding or lack of productive binding of clostridialneurotoxins thereby defines neuronal and non-neuronal cellsrespectively.

In addition to lacking high affinity binding sites for clostridialneurotoxins, absence of the correct internalisation and intracellularrouting mechanism, or additional factors that are not yet understood,would prevent clostridial neurotoxin action in non-neuronal cells.

It is known from WO96/33273 that hybrid clostridial neurotoxinsendopeptidases can be prepared and that these hybrids effectivelyinhibit release of neurotransmitters from neuronal cells to which theyare targeted, such as pain transmitting neurons. WO96/33273 describesthe activity of hybrids only in neuronal systems where neuronalmechanisms of internalisation and vesicular routing are operational.

Non-neuronal cells are, however, refractory to the effects ofclostridial neurotoxins, since simple application of clostridialneurotoxins to the surface of non-neuronal cells does not lead toinhibition of secretory vesicle exocytosis. This insensitivity ofnon-neuronal cells to clostridial neurotoxins may be due to absence ofthe requisite receptor, absence of the correct internalisation &intracellular routing mechanism, or additional factors that are not yetunderstood.

WO95/17904 describes the use of C. botulinum holotoxin in the treatmentof various disorders such as excessive sweating, lacrimation and mucussecretion, and pain. WO95/17904 describes treatment by targetingneuronal cells

It is an object of the present invention to provide methods andcompositions for inhibition of secretion from non-neuronal cells.

Accordingly, the present invention is based upon the use of acomposition which inhibits the exocytotic machinery in neuronal cellsand which surprisingly has been found to be effective at inhibitingexocytotic processes in non-neuronal cells.

A first aspect of the invention thus provides a method of inhibitingsecretion from a non-neuronal cell comprising administering an agentcomprising at least first and second domains, wherein the first domaincleaves one or more proteins essential to exocytosis and the seconddomain translocates the first domain into the cell.

Advantageously, the invention provides for inhibition of non-neuronalsecretion and enables treatment of disease caused, exacerbated ormaintained by such secretion.

An agent for use in the invention is suitably prepared by replacement ofthe cell-binding H_(C) domain of a clostridial neurotoxin with a ligandcapable of binding to the surface of non-neuronal cells. Surprisingly,this agent is capable of inhibiting the exocytosis of a variety ofsecreted substances from non-neuronal cells. By covalently linking aclostridial neurotoxin, or a hybrid of two clostridial neurotoxins, inwhich the H_(C) region of the H-chain has been removed or modified, to anew molecule or moiety, the Targeting Moiety (TM), an agent is producedthat binds to a binding site (BS) on the surface of the relevantnon-neuronal secretory cells. A further surprising aspect of the presentinvention is that if the L-chain of a clostridial neurotoxin, or afragment, variant or derivative of the L-chain containing theendopeptidase activity, is covalently linked to a TM which can alsoeffect internalisation of the L-chain, or a fragment of theendopeptidase activity, into the cytoplasm of a non-neuronal secretorycell, this also produces an agent capable of inhibiting secretion. Thus,the present invention overcomes the insusceptibility of non-neuronalcells to the inhibitory effects of clostridial neurotoxins.

An example of an agent of the invention is a polypeptide comprisingfirst and second domains, wherein said first domain cleaves one or morevesicle or plasma-membrane associated proteins essential to neuronalexocytosis and wherein said second domain translocates the polypeptideinto the cell or translocates at least that portion responsible for theinhibition of exocytosis into the non-neuronal cell. The polypeptide canbe derived from a neurotoxin in which case the polypeptide is typicallyfree of clostridial neurotoxin and free of any clostridial neurotoxinprecursor that can be converted into toxin by proteolytic action, beingaccordingly substantially non-toxic and suitable for therapeutic use.Accordingly, the invention may thus use polypeptides containing a domainequivalent to a clostridial toxin light chain and a domain providing thetranslocation function of the H_(N) of a clostridial toxin heavy chain,whilst lacking the functional aspects of a clostridial toxin H_(C)domain.

In use of the invention, the polypeptide is administered in vivo to apatient, the first domain is translocated into a non-neuronal cell byaction of the second domain and cleaves one or more vesicle orplasma-membrane associated proteins essential to the specific cellularprocess of exocytosis, and cleavage of these proteins results ininhibition of exocytosis, thereby resulting in inhibition of secretion,typically in a non-cytotoxic manner.

The polypeptide of the invention may be obtained by expression of arecombinant nucleic acid, preferably a DNA, and can be a singlepolypeptide, that is to say not cleaved into separate light and heavychain domains or two polypeptides linked for example by a disulphidebond.

The first domain preferably comprises a clostridial toxin light chain ora functional fragment or variant of a clostridial toxin light chain. Thefragment is optionally an N-terminal, or C-terminal fragment of thelight chain, or is an internal fragment, so long as it substantiallyretains the ability to cleave the vesicle or plasma-membrane associatedprotein essential to exocytosis. The minimal domains necessary for theactivity of the light chain of clostridial toxins are described in J.Biol. Chem., Vol. 267, No. 21, July 1992, pages 14721-14729. The varianthas a different peptide sequence from the light chain or from thefragment, though it too is capable of cleaving the vesicle orplasma-membrane associated protein. It is conveniently obtained byinsertion, deletion and/or substitution of a light chain or fragmentthereof. A variety of variants are possible, including (i) an N-terminalextension to a clostridial toxin light chain or fragment (ii) aclostridial toxin light chain or fragment modified by alteration of atleast one amino acid (iii) a C-terminal extension to a clostridial toxinlight chain or fragment, or (iv) combinations of 2 or more of (i)-(iii).In further embodiments of the invention, the variant contains an aminoacid sequence modified so that (a) there is no protease sensitive regionbetween the LC and H_(N) components of the polypeptide, or (b) theprotease sensitive region is specific for a particular protease. Thislatter embodiment is of use if it is desired to activate theendopeptidase activity of the light chain in a particular environment orcell, though, in general, the polypeptides of the invention are in anactive form prior to administration.

The first domain preferably exhibits endopeptidase activity specific fora substrate selected from one or more of SNAP-25, synaptobrevin/VAMP andsyntaxin. The clostridial toxin from which this domain can be obtainedor derived is preferably botulinum toxin or tetanus toxin. Thepolypeptide can further comprise a light chain or fragment or variant ofone toxin type and a heavy chain or fragment or variant of another toxintype.

The second domain preferably comprises a clostridial toxin heavy chainH_(N) portion or a fragment or variant of a clostridial toxin heavychain H_(N) portion. The fragment is optionally an N-terminal orC-terminal or internal fragment, so long as it retains the function ofthe H_(N) domain. Teachings of regions within the H_(N) responsible forits function are provided for example in Biochemistry 1995, 34, pages15175-15181 and Eur. J. Biochem, 1989, 185, pages 197-203. The varianthas a different sequence from the H_(N) domain or fragment, though ittoo retains the function of the H_(N) domain. It is convenientlyobtained by insertion, deletion and/or substitution of a H_(N) domain orfragment thereof, and examples of variants include (i) an N-terminalextension to a H_(N) domain or fragment, (ii) a C-terminal extension toa H_(N) domain or fragment, (iii) a modification to a H_(N) domain orfragment by alteration of at least one amino acid, or (iv) combinationsof 2 or more of (i)-(iii). The clostridial toxin is preferably botulinumtoxin or tetanus toxin.

In preparation of the polypeptides by recombinant means, methodsemploying fusion proteins can be employed, for example a fusion proteincomprising a fusion of (a) a polypeptide of the invention as describedabove with (b) a second polypeptide adapted for binding to achromatography matrix so as to enable purification of the fusion proteinusing said chromatography matrix. It is convenient for the secondpolypeptide to be adapted to bind to an affinity matrix, such asglutathione Sepharose, enabling rapid separation and purification of thefusion protein from an impure source, such as a cell extract orsupernatant.

One second purification polypeptide is glutathione-S-transferase (GST),and others may be chosen so as to enable purification on achromatography column according to conventional techniques.

In a second aspect of the invention there is provided a method ofinhibiting secretion from selected non-neuronal cells responsible forregulated secretion by administering an agent of the invention.

In a third aspect of the invention there is provided a method oftreatment of disease resulting, or caused or maintained by secretionsfrom non-neuronal cells, comprising administering an agent of theinvention.

In further aspects of the invention there are provided agents of theinvention targeted to non-neuronal cells responsible for secretion.

In one embodiment of the invention, an agent is provided for thetreatment of conditions resulting from hyperfunction of endocrine cells,for example endocrine neoplasia.

Accordingly, an agent of the invention is used in the treatment ofendocrine hyperfunction, to inhibit secretion of endocrine cell-derivedchemical messengers. An advantage of the invention is that effectivetreatment of endocrine hyperfunction and associated disease states isnow provided, offering relief to sufferers where hitherto there was noneand no such agent available.

A further advantage of the invention is that agents are made availablewhich, in use, result in the inhibition of or decrease in hypersecretionof multiple hormones from a single endocrine gland. Thus, the multitudeof disorders that result from hyperfunction of one gland (e.g. theanterior pituitary) will be simultaneously treated by a reduction in thefunction of the hypersecreting gland.

The agent preferably comprises a ligand or targeting domain which bindsto an endocrine cell, and is thus rendered specific for these celltypes. Examples of suitable ligands include iodine; thyroid stimulatinghormone (TSH); TSH receptor antibodies; antibodies to the islet-specificmonosialo-ganglioside GM2-1; insulin, insulin-like growth factor andantibodies to the receptors of both; TSH releasing hormone (protirelin)and antibodies to its receptor; FSH/LH releasing hormone (gonadorelin)and antibodies to its receptor; corticotrophin releasing hormone (CRH)and antibodies to its receptor; and ACTH and antibodies to its receptor.According to the invention, an endocrine targeted agent may thus besuitable for the treatment of a disease selected from: endocrineneoplasia including MEN; thyrotoxicosis and other diseases dependent onhypersecretions from the thyroid; acromegaly, hyperprolactinaemia,Cushings disease and other diseases dependent on anterior pituitaryhypersecretion; hyperandrogenism, chronic anovulation and other diseasesassociated with polycystic ovarian syndrome.

In a further embodiment, an agent of the invention is used for thetreatment of conditions resulting from secretions of inflammatory cells,for example allergies. Ligands suitable to target agent to these cellsinclude (i) for mast cells, complement receptors in general, includingC4 domain of the Fc IgE, and antibodies/ligands to the C3a/C4a-Rcomplement receptor; (ii) for eosinophils, antibodies/ligands to theC3a/C4a-R complement receptor, anti VLA-4 monoclonal antibody, anti-IL5receptor, antigens or antibodies reactive toward CR4 complementreceptor; (iii) for macrophages and monocytes, macrophage stimulatingfactor, (iv) for macrophages, monocytes and neutrophils, bacterial LPSand yeast B-glucans which bind to CR3, (v) for neutrophils, antibody toOX42, an antigen associated with the iC3b complement receptor, or IL8;(vi) for fibroblasts, mannose 6-phosphate/insulin-like growthfactor-beta (M6P/IGF-II) receptor and PA2.26, antibody to a cell-surfacereceptor for active fibroblasts in mice.

According to a preferred embodiment of the present invention, the TM isa growth factor, preferably an epidermal growth factor (EGF), vascularendothelial growth factor, platelet-derived growth factor, keratinocytegrowth factor, hepatocyte growth factor, transforming growth factoralpha, transforming growth factor beta.

According to another preferred embodiment of the present invention, theTM is a peptide or protein that binds to an inflammatory cell. Apreferred example of such a TM is an integrin-binding protein.

Integrins are obligate heterodimer transmembrane proteins containing twodistinct chains a (alpha) and D (beta) subunits. In mammals, 19 alphaand 8 beta subunits have been characterised—see Humphries, M. J. (2000),Integrin structure. Biochem Soc Trans. 28: 311-339, which is hereinincorporated by reference thereto. Integrin subunits span through theplasma membrane, and in general have very short cytoplasmic domains ofabout 40-70 amino acids. Outside the cell plasma membrane, the alpha andbeta chains lie close together along a length of about 23 nm, the final5 nm NH₂-termini of each chain forming a ligand-binding region to whichan agent of the present invention binds.

Preferred integrin-binding proteins of the present invention comprisethe amino sequence Arg-Gly-Asp (“RGD”), which binds to theabove-described ligand-binding region—see Craig. D et al. (2004),Structural insights into how the MIDAS ion stabilizes integrin bindingto an RGD peptide under force. Structure, vol. 12, pp 2049-2058, whichis herein incorporated by reference thereto.

In one embodiment, the integrin-binding protein TMs of the presentinvention have an amino acid length of between 3 and 100, preferablybetween 3 and 50, more preferably between 5 and 25, and particularlypreferably between 5 and 15 amino acid residues.

The TMs of the present invention may form linear or cyclic structures.

Preferred integrin-binding TMs of the present invention include actin,alpha-actinin, focal contact adhesion kinase, paxillin, talin, RACK1,collagen, laminin, fibrinogen, heparin, phytohaemagglutinin,fibronectin, vitronectin, VCAM-1, ICAM-1, ICAM-2 and serum protein. Manyintegrins recognise the triple Arg-Gly-Asp (RGD) peptide sequence(Ruoslahti, 1996). The RGD motif is found in over 100 proteins includingfibronectin, tenascin, fibrinogen and vitronectin. The RGD-integrininteraction is exploited as a conserved mechanism of cell entry by manypathogens including coxsackievirus (Roivaninen et al., 1991) andadenovirus (Mathias et al., 1994).

Additionally preferred integrin-binding TMs of the present inventioninclude proteins selected from the following sequences:Arg-Gly-Asp-Phe-Val (SEQ ID NO:23); Arg-Gly-Asp-{D-Phe}-{N-methyl-Val}(SEQ ID NO:23); RGDFV (SEQ ID NO:23); RGDfNMeV (SEQ ID NO:23); GGRGDMFGA(SEQ ID NO:21); GGCRGDMFGCA (SEQ ID NO:22); GRGDSP (SEQ ID NO:26);GRGESP (SEQ ID NO:27); PLAEIDGIEL (SEQ ID NO:24 and CPLAEIDGIELC (SEQ IDNO:25). Reference to the above sequences embraces linear and cyclicforms, together with peptides exhibiting at least 80%, 85%, 90%, 95%,98%, 99% sequence identity with said sequences. All of said TMspreferably retain the “RGD” tri-peptide sequence.

Diseases thus treatable according to the invention include diseasesselected from allergies (seasonal allergic rhinitis (hay fever),allergic conjunctivitis, vasomotor rhinitis and food allergy),eosinophilia, asthma, rheumatoid arthritis, systemic lupuserythematosus, discoid lupus erythematosus, ulcerative colitis, Crohn'sdisease, hemorrhoids, pruritus, glomerulonephritis, hepatitis,pancreatitis, gastritis, vasculitis, myocarditis, psoriasis, eczema,chronic radiation-induced fibrosis, lung scarring and other fibroticdisorders.

VAMP expression has been demonstrated in B-lymphocytes [see Olken, S. K.and Corley, R. B. 1998, Mol. Biol. Cell. 9, 207a]. Thus, an agentaccording to the present invention, when targeted to a B-lymphocyte andfollowing internalisation and retrograde transport, may exert itsinhibitory effect on such target cells.

In a further embodiment, an agent of the invention is provided for thetreatment of conditions resulting from secretions of the exocrine cells,for example acute pancreatitis (Hansen et al, 1999, J. Biol. Chem. 274,22871-22876). Ligands suitable to target agent to these cells includepituitary adenyl cyclase activating peptide (PACAP-38) or an antibody toits receptor. The present invention also concerns treatment of mucushypersecretion from mucus-secreting cells located in the alimentarytract, in particular located in the colon.

Gaisano, H. Y. et al. (1994) J. Biol. Chem. 269, pp. 17062-17066 hasdemonstrated that, following in vitro membrane permeabilisation topermit cellular entry, tetanus toxin light chain cleaves avesicle-associated membrane protein (VAMP) isoform 2 in rat pancreaticzymogen granules, and inhibits enzyme secretion. Thus, an agentaccording to the present invention, when targeted to a pancreatic celland following internalisation and retrograde transport, may exert itsinhibitory effect on such target cells.

In a further embodiment, an agent of the invention is used for thetreatment of conditions resulting from secretions of immunologicalcells, for example autoimmune disorders where B lymphocytes are to betargeted (immunosuppression). Ligands suitable to target agent to thesecells include Epstein Barr virus fragment/surface feature or idiotypicantibody (binds to CR2 receptor on B-lymphocytes and lymph nodefollicular dendritic cells). Diseases treatable include myastheniagravis, rheumatoid arthritis, systemic lupus erythematosus, discoidlupus erythematosus, organ transplant, tissue transplant, fluidtransplant, Graves disease, thyrotoxicosis, autoimmune diabetes,hemolytic anaemia, thrombocytopenic purpura, neutropenia, chronicautoimmune hepatitis, autoimmune gastritis, pernicious anaemia,Hashimoto's thyroiditis, Addison's disease, Sjogren's syndrome, primarybiliary cirrhosis, polymyositis, scleroderma, systemic sclerosis,pemphigus vulgaris, bullous pemphigoid, myocarditis, rheumatic carditis,glomerulonephritis (Goodpasture type), uveitis, orchitis, ulcerativecolitis, vasculitis, atrophic gastritis, pernicious anaemia, type 1diabetes mellitus.

By using cell permeabilisation techniques it has been possible tointernalise BoNT/C into eosinophils [see Pinxteren J A, et al (2000)Biochimie, April; 82(4):385-93 Thirty years of stimulus-secretioncoupling: from Ca(2⁺) to GTP in the regulation of exocytosis]. Followinginternalisation, BoNT/C exerted an inhibitory effect on exocytosis ineosinophils. Thus, an agent according to the present invention, whentargeted to an eosinophil and following internalisation and retrogradetransport, may exert its inhibitory effect on such target cells.

In a further embodiment of the invention, an agent is provided for thetreatment of conditions resulting from secretions of cells of thecardiovascular system. Suitable ligands for targeting platelets for thetreatment of disease states involving inappropriate platelet activationand thrombus formation include thrombin and TRAP (thrombin receptoragonist peptide) or antibodies to CD31/PECAM-1, CD24 or CD106NCAM-1, andligands for targeting cardiovascular endothelial cells for the treatmentof hypertension include GP1b surface antigen recognising antibodies.

In a further embodiment of the invention, an agent is provided for thetreatment of bone disorders. Suitable ligands for targeting osteoblastsfor the treatment of a disease selected from osteopetrosis andosteoporosis include calcitonin, and for targeting an agent toosteoclasts include osteoclast differentiation factors (eg. TRANCE, orRANKL or OPGL), and an antibody to the receptor RANK.

In use of the invention, a Targeting moiety (TM) provides specificityfor the BS on the relevant non-neuronal secretory cells. The TMcomponent of the agent can comprise one of many cell binding molecules,including, but not limited to, antibodies, monoclonal antibodies,antibody fragments (Fab, F(ab)′₂, Fv, ScFv, etc.), lectins, hormones,cytokines, growth factors, peptides, carbohydrates, lipids, glycons,nucleic acids or complement components.

The TM is selected in accordance with the desired cell-type to which theagent of the present invention is to be targeted, and preferably has ahigh specificity and/or affinity for non-neuronal target cells.Preferably, the TM does not substantially bind to neuronal cells of thepresynaptic muscular junction, and thus the agent is substantiallynon-toxic in that it is not capable of effecting muscular paralysis.This is in contrast to clostridial holotoxin which targets thepresynaptic muscular junction and effects muscular paralysis. Inaddition, preferably the TM does not substantially bind to neuronalperipheral sensory cells, and thus the agent does not exert anysubstantial analgesic effect. Preferably, the TM does not substantiallybind to neuronal cells, and does not therefore permit the agent to exertan inhibitory effect on secretion in a neuronal cell.

It is known in the art that the H_(C) portion of the neurotoxin moleculecan be removed from the other portion of the H-chain, known as H_(N),such that the H_(N) fragment remains disulphide linked to the L-chain ofthe neurotoxin providing a fragment known as LH_(N). Thus, in oneembodiment of the present invention the LH_(N) fragment of a clostridialneurotoxin is covalently linked, using linkages which may include one ormore spacer regions, to a TM.

In another embodiment of the invention, the H_(C) domain of aclostridial neurotoxin is mutated, blocked or modified, e.g. by chemicalmodification, to reduce or preferably incapacitate its ability to bindthe neurotoxin to receptors at the neuromuscular junction. This modifiedclostridial neurotoxin is then covalently linked, using linkages whichmay include one or more spacer regions, to a TM.

In another embodiment of the invention, the heavy chain of a clostridialneurotoxin, in which the H_(C) domain is mutated, blocked or modified,e.g. by chemical modification, to reduce or preferably incapacitate itsability to bind the neurotoxin to receptors at the neuromuscularjunction, is combined with the L-chain of a different clostridialneurotoxin. This hybrid, modified clostridial neurotoxin is thencovalently linked, using linkages which may include one or more spacerregions, to a TM.

In another embodiment of the invention, the H_(N) domain of aclostridial neurotoxin is combined with the L-chain of a differentclostridial neurotoxin. This hybrid LH_(N) is then covalently linked,using linkages which may include one or more spacer regions, to a TM.

In another embodiment of the invention, the light chain of a clostridialneurotoxin, or a fragment of the light chain containing theendopeptidase activity, is covalently linked, using linkages which mayinclude one or more spacer regions, to a TM which can also effect theinternalisation of the L-chain, or a fragment of the L-chain containingthe endopeptidase activity, into the cytoplasm of the relevantnon-neuronal cells responsible for secretion.

In another embodiment of the invention, the light chain of a clostridialneurotoxin, or a fragment of the light chain containing theendopeptidase activity, is covalently linked, using linkages which mayinclude one or more spacer regions, to a translocation domain to effecttransport of the endopeptidase fragment into the cytosol. Examples oftranslocation domains derived from bacterial neurotoxins are as follows:

Botulinum type A neurotoxin—amino acid residues (449-871)

Botulinum type B neurotoxin—amino acid residues (441-858)

Botulinum type C neurotoxin—amino acid residues (442-866)

Botulinum type D neurotoxin—amino acid residues (446-862)

Botulinum type E neurotoxin—amino acid residues (423-845)

Botulinum type F neurotoxin—amino acid residues (440-864)

Botulinum type G neurotoxin—amino acid residues (442-863)

Tetanus neurotoxin—amino acid residues (458-879)

other clostridial sources include—C. butyricum, and C. argentinense.

[for the genetic basis of toxin production in Clostridium botulinum andC. tetani, see Henderson et al (1997) in The Clostridia: MolecularBiology and Pathogenesis, Academic press].

In addition to the above translocation domains derived from clostridialsources, other non-clostridial sources may be employed in an agentaccording to the present invention. These include, for example,diphtheria toxin [London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51], Pseudomonas exotoxin A [Prior et al (1992) Biochem., 31, pp.3555-3559], influenza virus haemagglutinin fusogenic peptides [Wagner etal (1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata etal (1992) Biochem., 31, pp. 1986-1992].

In use, the domains of an agent according to the present invention areassociated with each other. In one embodiment, two or more of theDomains may be joined together either directly (e.g. by a covalentlinkage), or via a linker molecule. Conjugation techniques suitable foruse in the present invention have been well documented:—Chemistry ofprotein conjugation and cross-linking. Edited by Wong, S. S. 1993, CRCPress Inc., Florida; and Bioconjugate techniques, Edited by Hermanson,G. T. 1996, Academic Press, London, UK.

Direct linkage of two or more of Domains is now described with referenceto clostridial neurotoxins and to the present Applicant's nomenclatureof clostridial neurotoxin domains, namely Domain B (contains the bindingdomain), Domain T (contains the translocation domain) and Domain E(contains the protease domain), although no limitation thereto isintended.

In one embodiment of the present invention, Domains E and T may be mixedtogether in equimolar quantities under reducing conditions andcovalently coupled by repeated dialysis (e.g. at 4° C., with agitation),into physiological salt solution in the absence of reducing agents. Atthis stage, in contrast to Example 6 of WO94/21300, the E-T complex isnot blocked by iodoacetamide, therefore any remaining free —SH groupsare retained.

Domain B is then modified, for example, by derivatisation with SPDPfollowed by subsequent reduction. In this reaction, SPDP does not remainattached as a spacer molecule to Domain B, but simply increases theefficiency of this reduction reaction.

Reduced domain B and the E-T complex may then be mixed undernon-reducing conditions (e.g. at 4° C.) to form a disulphide-linkedE-T-B “agent”.

In another embodiment, a coupled E-T complex may be prepared accordingto Example 6 of WO94/21300, including the addition of iodoacetamide toblock free sulphydryl groups. However, the E-T complex is not furtherderivatised, and the remaining chemistry makes use of the free amino(—NH₂) groups on amino acid side chains (e.g. lysine, and arginine aminoacids).

Domain B may be derivatised using carbodiimide chemistry (e.g. usingEDC) to activate carboxyl groups on amino acid side chains (e.g.glutamate, and aspartate amino acids), and the E-T complex mixed withthe derivatised Domain B to result in a covalently coupled (amide bond)E-T-B complex.

Suitable methodology for the creation of such an agent is, for example,as follows:—

Domain B was dialysed into MES buffer (0.1 M MES, 0.1 M sodium chloride,pH 5.0) to a final concentration of 0.5 mg/ml. EDAC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) was addedto final concentrations of 0.2 mg/ml and reacted for 30 min at roomtemperature. Excess EDAC was removed by desalting over a MES bufferequilibrated PD-10 column (Pharmacia). The derivatised domain B wasconcentrated (to >2 mg/ml) using Millipore Biomax 10 concentrators. TheE-T complex (1 mg/ml) was mixed for 16 hours at 4° C., and the E-T-Bcomplex purified by size-exclusion chromatography over a Superose 12HR10/30 column (Pharmacia) to remove unreacted Domain B (column buffer:50 mM sodium phosphate pH6.5+20 mM NaCl).

As an alternative to direct covalent linkage of the various Domains ofan agent according to the present invention, suitable spacer moleculesmay be employed. The term linker molecule is used synonymously withspacer molecule. Spacer technology was readily available prior to thepresent application.

For example, one particular coupling agent (SPDP) is described inExample 6 of WO94/21300 (see lines 3-5 on page 16). In Example 6, SPDPis linked to an E-T complex, thereby providing an E-T complex includinga linker molecule. This complex is then reacted a Domain B, whichbecomes attached to the E-T complex via the linker molecule. In thismethod, SPDP results in a spacing region of approximately 6.8 Angstromsbetween different Domains of the “agent” of the present invention.

A variant of SPDP known as LC-SPDP is identical in all respects to SPDPbut for an increased chain length. LC-SPDP may be used to covalentlylink two Domains of the “agent” of the present invention resulting in a15.6 Angstrom spacing between these Domains.

Examples of spacer molecules include, but are not limited to:—

(GGGGS)₂ (SEQ ID NO:28), elbow regions of Fab—[see Anand et al., (1991)J. Biol. Chem. 266, 21874-9];

(GGGGS)₃ (SEQ ID NO:28)-[see Brinkmann et al. (1991) Proc. Natl. Acad.Sci. 88, 8616-20];

the interdomain linker of cellulose—[see Takkinen et al. (1991) ProteinEng, 4, 837-841];

PPPIEGR (SEQ ID NO:29)-[see Kim (1993) Protein Science, 2, 348-356];

Collagen-like spacer—[see Rock (1992) Protein Engineering, vol 5, No 6,pp 583-591]; and

Trypsin-sensitive diphtheria toxin peptide-[see O'Hare (1990) FEBS, vol273, No 1, 2, pp 200-204].

In a further embodiment of the present invention, an agent having thestructure E-X-T-X-B, where “X” is a spacer molecule between each domain,may be prepared, for example, as follows:—

Domain E is derivatised with SPDP, but not subsequently reduced. Thisresults in an SPDP-derivatised Domain E.

Domain T is similarly prepared, but subsequently reduced with 10 mMdithiothreitol (DTT). The 10 mM DTT present in the Domain T preparation,following elution from the QAE column (see Example 6 in WO94/21300), isremoved by passage of Domain T through a sephadex G-25 columnequilibrated in PBS.

Domain T free of reducing agent is then mixed with the SPDP-derivatisedDomain E, with agitation at 4° C. for 16 hours. E-T complex is isolatedfrom free Domain E and from free Domain T by size-exclusionchromatography (Sephadex G-150). Whereafter, the same procedure can befollowed as described in Example 6 of WO94/21300 for rederivatisation ofthe E-T complex with SPDP, and subsequent coupling thereof to the freesulphydryl on Domain B.

The agents according to the present invention may be preparedrecombinantly.

In one embodiment, the preparation of a recombinant agent may involvearrangement of the coding sequences of the selected TM and clostridialneurotoxin component in a single genetic construct. These codingsequences may be arranged in-frame so that subsequent transcription andtranslation is continuous through both coding sequences and results in afusion protein. All constructs would have a 5′ ATG codon to encode anN-terminal methionine, and a C-terminal translational stop codon.

Thus, a the light chain of a clostridial neurotoxin (or a fragment ofthe light chain containing the endopeptidase activity) may be expressedrecombinantly as a fusion protein with a TM which can also effect theinternalisation of the L-chain (or a fragment thereof) into thecytoplasm of the relevant non-neuronal cells responsible for secretion.The expressed fusion protein may also include one or more spacerregions.

In the case of an agent based on clostridial neurotoxin, the followinginformation would be required to produce said agent recombinantly:—(i)DNA sequence data relating to a selected TM; (ii) DNA sequence datarelating to the clostridial neurotoxin component; and (iii) a protocolto permit construction and expression of the construct comprising (i)and (ii).

All of the above basic information (i)-(iii) are either readilyavailable, or are readily determinable by conventional methods. Forexample, both WO98/07864 and WO99/17806 exemplify clostridial neurotoxinrecombinant technology suitable for use in the present application.

In addition, methods for the construction and expression of theconstructs of the present invention may employ information from thefollowing references and others:

Lorberboum-Galski, H., FitzGerald, D., Chaudhary, V., Adhya, S., Pastan,I. (1988). Cytotoxic activity of an interleukin 2-Pseudomonas exotoxinchimeric protein produced in Escherichia coli. Proc Natl Acad Sci USA85(6):1922-6;

Murphy, J. R. (1988) Diphtheria-related peptide hormone gene fusions: amolecular genetic approach to chimeric toxin development. Cancer TreatRes; 37:123-40;

Williams, D. P., Parker, K., Bacha, P., Bishai, W., Borowski, M.,Genbauffe, F., Strom, T. B., Murphy, J. R. (1987). Diphtheria toxinreceptor binding domain substitution with interleukin-2: geneticconstruction and properties of a diphtheria toxin-related interleukin-2fusion protein. Protein Eng; 1(6):493-8;

Arora, N., Williamson, L. C., Leppla, S. H., Halpern, J. L. (1994).Cytotoxic effects of a chimeric protein consisting of tetanus toxinlight chain and anthrax toxin lethal factor in non-neuronal cells J BiolChem, 269(42):26165-71;

Brinkmann, U., Reiter, Y., Jung, S. H., Lee, B., Pastan, I. (1993). Arecombinant immunotoxin containing a disulphide-stabilized Fv fragment.Proc Natl Acad Sci USA; 90(16):7538-42; and

O'Hare, M., Brown, A. N., Hussain, K., Gebhardt, A., Watson, G.,Roberts, L. M., Vitetta, E. S., Thorpe, P. E., Lord, J. M. (1990).Cytotoxicity of a recombinant ricin-A-chain fusion protein containing aproteolytically-cleavable spacer sequence. FEBS Lett October 29;273(1-2):200-4.

Suitable clostridial neurotoxin sequence information relating to L- andLH_(N)-chains may be obtained from, for example, Kurazono, H. (1992) J.Biol. Chem., vol. 267, No. 21, pp. 14721-14729; and Popoff, M. R., andMarvaud, J.-C. (1999) The Comprehensive Sourcebook of Bacterial ProteinToxins, 2nd edition (ed. Alouf, J. E., and Freer, J. H.), AcademicPress, pp. 174-201.

Similarly, suitable TM sequence data are widely available in the art.Alternatively, any necessary sequence data may be obtained by techniqueswhich were well-known to the skilled person.

For example, DNA encoding the TM component may be cloned from a sourceorganism by screening a cDNA library for the correct coding region (forexample by using specific oligonucleotides based on the known sequenceinformation to probe the library), isolating the TM DNA, sequencing thisDNA for confirmation purposes, and then placing the isolated DNA in anappropriate expression vector for expression in the chosen host.

As an alternative to isolation of the sequence from a library, theavailable sequence information may be employed to prepare specificprimers for use in PCR, whereby the coding sequence is then amplifieddirectly from the source material and, by suitable use of primers, maybe cloned directly into an expression vector.

Another alternative method for isolation of the coding sequence is touse the existing sequence information and synthesise a copy, possiblyincorporating alterations, using DNA synthesis technology. For example,DNA sequence data may be generated from existing protein and/or RNAsequence information. Using DNA synthesis technology to do this (and thealternative described above) enables the codon bias of the codingsequence to be modified to be optimal for the chosen expression host.This may give rise to superior expression levels of the fusion protein.

Optimisation of the codon bias for the expression host may be applied tothe DNA sequences encoding the TM and clostridial components of theconstruct. Optimisation of the codon bias is possible by application ofthe protein sequence into freely available DNA/protein databasesoftware, e.g. programs available from Genetics Computer Group, Inc.

According to a further aspect of the present invention, nucleic acidencoding the light chain of a clostridial neurotoxin (or a fragment ofthe light chain containing the endopeptidase activity), may beassociated with a TM which can also effect the internalisation of thenucleic acid encoding the L-chain (or a fragment thereof) into thecytoplasm of the relevant non-neuronal cells responsible for secretion.The nucleic acid sequence may be coupled to a translocation domain, andoptionally to a targeting moiety, by for example direct covalent linkageor via spacer molecule technology. Ideally, the coding sequence will beexpressed in the target cell.

Thus, the agent of the present invention may be the expression productof a recombinant gene delivered independently to the preferred site ofaction of the agent. Gene delivery technologies are widely reported inthe literature [reviewed in “Advanced Drug Delivery Reviews” Vol. 27,(1997), Elsevier Science Ireland Ltd].

According to another aspect, the present invention therefore provides amethod of treating a condition or disease which is susceptible oftreatment with a nucleic acid in a mammal e.g. a human which comprisesadministering to the sufferer an effective, non-toxic amount of acompound of the invention. A condition or disease which is susceptibleof treatment with a nucleic acid may be for example a condition ordisease which may be treated by or requiring gene therapy. The preferredconditions or diseases susceptible to treatment according to the presentinvention, together with the preferred TMs, have been describedpreviously in this specification. Similarly, the preferred first domainswhich cleave one or more proteins (eg. SNAP-25, synaptobrevin andsyntaxin) essential to exocytosis have been described previously in thisspecification. The various domains of an agent for use in gene therapymay be directly linked (e.g. via a covalent bond) or indirectly linked(e.g. via a spacer molecule), as for example previously described inthis specification.

The invention further provides a compound of the invention for use as anactive therapeutic substance, in particular for use in treating acondition or disease as set forth in the present claims.

The invention further provides pharmaceutical compositions comprising anagent or a conjugate of the invention and a pharmaceutically acceptablecarrier.

In use the agent or conjugate will normally be employed in the form of apharmaceutical composition in association with a human pharmaceuticalcarrier, diluent and/or excipient, although the exact form of thecomposition will depend on the mode of administration.

The conjugate may, for example, be employed in the form of an aerosol ornebulisable solution for inhalation or a sterile solution for parenteraladministration, intra-articular administration or intra-cranialadministration.

For treating endocrine targets, i.v. injection, direct injection intogland, or aerosolisation for lung delivery are preferred; for treatinginflammatory cell targets, i.v. injection, sub-cutaneous injection, orsurface patch administration are preferred; for treating exocrinetargets, i.v. injection, or direct injection into the gland arepreferred; for treating immunological targets, i.v. injection, orinjection into specific tissues e.g. thymus, bone marrow, or lymphtissue are preferred; for treatment of cardiovascular targets, i.v.injection is preferred; and for treatment of bone targets, i.v.injection, or direct injection is preferred. In cases of i.v. injection,this should also include the use of pump systems.

The dosage ranges for administration of the compounds of the presentinvention are those to produce the desired therapeutic effect. It willbe appreciated that the dosage range required depends on the precisenature of the conjugate, the route of administration, the nature of theformulation, the age of the patient, the nature, extent or severity ofthe patient's condition, contraindications, if any, and the judgement ofthe attending physician.

Suitable daily dosages are in the range 0.0001-1 mg/kg, preferably0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularlypreferably 0.004-0.5 mg/kg. The unit dosage can vary from less that 1microgram to 30 mg, but typically will be in the region of 0.01 to 1 mgper dose, which may be administered daily or less frequently, such asweekly or six monthly.

Wide variations in the required dosage, however, are to be expecteddepending on the precise nature of the conjugate, and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection.

Variations in these dosage levels can be adjusted using standardempirical routines for optimisation, as is well understood in the art.

Compositions suitable for injection may be in the form of solutions,suspensions or emulsions, or dry powders which are dissolved orsuspended in a suitable vehicle prior to use.

Fluid unit dosage forms are typically prepared utilising a pyrogen-freesterile vehicle. The active ingredients, depending on the vehicle andconcentration used, can be either dissolved or suspended in the vehicle.

Solutions may be used for all forms of parenteral administration, andare particularly used for intravenous injection. In preparing solutionsthe compound can be dissolved in the vehicle, the solution being madeisotonic if necessary by addition of sodium chloride and sterilised byfiltration through a sterile filter using aseptic techniques beforefilling into suitable sterile vials or ampoules and sealing.Alternatively, if solution stability is adequate, the solution in itssealed containers may be sterilised by autoclaving.

Advantageously additives such as buffering, solubilising, stabilising,preservative or bactericidal, suspending or emulsifying agents and/orlocal anaesthetic agents may be dissolved in the vehicle.

Dry powders which are dissolved or suspended in a suitable vehicle priorto use may be prepared by filling pre-sterilised drug substance andother ingredients into a sterile container using aseptic technique in asterile area.

Alternatively the agent and other ingredients may be dissolved in anaqueous vehicle, the solution is sterilized by filtration anddistributed into suitable containers using aseptic technique in asterile area. The product is then freeze dried and the containers aresealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous orintradermal injection, are prepared in substantially the same manner,except that the sterile compound is suspended in the sterile vehicle,instead of being dissolved and sterilisation cannot be accomplished byfiltration. The compound may be isolated in a sterile state oralternatively it may be sterilised after isolation, e.g. by gammairradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone isincluded in the composition to facilitate uniform distribution of thecompound.

Compositions suitable for administration via the respiratory tractinclude aerosols, nebulisable solutions or microfine powders forinsufflation. In the latter case, particle size of less than 50 microns,especially less than 10 microns, is preferred. Such compositions may bemade up in a conventional manner and employed in conjunction withconventional administration devices.

The agent described in this invention can be used in vivo, eitherdirectly or as a pharmaceutically acceptable salt, for the treatment ofconditions involving secretion from non-neuronal cells, such ashypersecretion of endocrine cell derived chemical messengers,hypersecretion from exocrine cells, secretions from the cells of theimmune system, the cardiovascular system and from bone cells.

The present invention will now be described by reference to thefollowing examples illustrated by the accompanying drawings in which:—

FIG. 1 shows SDS-PAGE analysis of WGA-LH_(N)/A purification scheme;

FIG. 2 shows activity of WGA-LH_(N)/A on release of transmitter fromHIT-T15 cells;

FIG. 3 shows correlation of SNAP-25 cleavage with inhibition ofneurotransmitter release following application of WGA-LH_(N)/A toHIT-T15 cells;

FIG. 4 shows activity of WGA-LH_(N)/A on release of [³H]-noradrenalinefrom undifferentiated PC12 cells;

FIG. 5 shows a Western blot indicating expression of recLH_(N)/B in E.coli;

FIG. 6 shows in vitro cleavage of synthetic VAMP peptide by recLH_(N)/B;

FIG. 7 shows the effect of low pH and BoNT/B treatment on stimulated vonWillebrands Factor (vWF) release from human umbilical vein endothelialcells;

FIG. 8 shows release of [³H]-glucosamine labelled high molecular weightmaterial from LS180 cells;

FIG. 9 shows the effect of low pH and BoNT/B treatment on stimulated.beta.-glucuronidase release from differentiated HL60 cells;

FIG. 10 shows purification of a LHN/C-EGF fusion protein;

FIG. 11 shows purification of a LHN/B-EGF fusion protein;

FIG. 12 shows purification of a LHN/C-RGD fusion protein;

FIG. 13 shows purification of a LHN/C-cyclic RGD fusion protein;

FIG. 14 shows purification of a LC/C-RGD-HN/C fusion protein;

FIG. 15 shows VAMP cleavage activity of LHN/B-EGF;

FIG. 16 shows effect of 10 nm Syntaxin compounds con LPS-mediated IL-8secretion by THP-1 cells;

FIG. 17 shows effect of 10 nm Syntaxin compounds con LPS-mediated IL-10secretion by RPMI-8226 cells;

FIG. 18 shows effect of EGF and fusions on IL-8 production and onLPS-stimulated IL-8 secretion; and

FIG. 19 shows effect of EGF and fusions on IP-10 production and onPHA-stimulated IP-10 secretion.

FIGS. 5-19 are now described in more detail.

Referring to FIG. 5, MBP-LH_(N)/B was expressed in E. coli as describedin Example 4. Lane 1 represents the profile of the expressed fusionprotein in E. coli. Lane 2 represents the profile of fusion proteinexpression in the crude E. coli lysate. Lane 3 represents the profile ofthe MBP-LH_(N)/B following purification by immobilised amylose.Molecular weights in kDa are indicated to the right side of the Figure.

Referring to FIG. 6, dilutions of recLH_(N)/B (prepared as described inExample 4) and BoNT/B were compared in an in vitro peptide cleavageassay. Data indicate that the recombinant product has similar catalyticactivity to that of the native neurotoxin, indicating that therecombinant product has folded correctly into an active conformation.

Referring to FIG. 7, cells were exposed to pH 4.7 media with or without500 nM BoNT/B (control cells received pH7.4 medium) for 2.5 hours thenwashed. 24 hours later release of vWF was stimulated using 1 mMhistamine and the presented results are the net stimulated release withbasal subtracted. Results are presented in mIU of vWF/ml and are themean +/−SEM of three determinations apart from pH 4.7 alone which is twodeterminations. pH 4.7+BoNT/B has reduced vWF release by 27.4% comparedto pH 4.7 controls.

Referring to FIG. 8, high molecular weight mucin synthesising coloncarcinoma LS180 cells were treated with pH 4.7 medium and pH 4.7 mediumcontaining 500 nM botulinum neurotoxin type B (BoNT/B) for four hoursthen labelled with [³H]-glucosamine for 18 hours. Release of highmolecular weight material was stimulated with 10 μM ionomycin and[³H]-glucosamine labelled material recovered by ultracentrifugation andcentrifugal molecular weight sieving. Radiolabel of release of labelledhigh molecular weight material was determined by scintillation countingand net stimulated release calculated by subtracting non-stimulatedbasal values. Data are expressed as disintegrations per minute(dpm)+/−SEM of three determinations. BoNT/B co-treatment clearlyinhibits the release of high molecular weight material from these mucinsynthesising cells and in this experiment a 74.5% reduction was seen.

Referring to FIG. 9, cells were exposed to pH 4.8 media with or without500 nM BoNT/B (control cells received pH 7.4 medium) for 2.5 hours thenwashed and differentiated for 40 hours by the addition of 300 μMdibutyryl cyclic AMP (dbcAMP). Cells were stimulated with fMet-Leu-Phe(1 μM)+ATP (100 μM) in the presence of cytochalasin B (5 μM) for 10minutes and released β-glucuronidase determined by colourimetric assay.Net stimulated release was calculated by subtraction of unstimulatedbasal release values from stimulated values and released activity isexpressed as a percentage of the total activity present in the cells.Data are the mean +/−SEM of three determinations. BoNT/B treatment inlow pH medium significantly inhibited stimulated release ofβ-glucuronidase compared to cells treated with low pH alone (p=0.0315when subjected to a 2 tailed Student T test with groups of unequalvariance).

Referring to FIG. 10, using the methodology outlined in Example 11, aLHN/C-EGF fusion protein was purified from E. coli BL21 cells. Briefly,the soluble products obtained following cell disruption were applied toa nickel-charged affinity capture column. Bound proteins were elutedwith 100 mM imidazole, treated with Factor Xa to activate the fusionprotein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. Lane 1 & 6:Molecular mass markers (kDa), lane 2: Clarified crude cell lysate, lane3: First nickel chelating Sepharose column eluant, lane 4: Factor Xadigested protein, lane 5: Purified LHN/C-EGF under non-reducingconditions, lane 7: Purified LHN/C-EGF under reduced conditions.

Referring to FIG. 11, using the methodology outlined in Example 12, aLHN/B-EGF fusion protein was purified from E. coli BL21 cells. Briefly,the soluble products obtained following cell disruption were applied toa nickel-charged affinity capture column. Bound proteins were elutedwith 100 mM imidazole, treated with Factor Xa and enterokinase toactivate the fusion protein and remove the maltose-binding protein (MBP)tag, then re-applied to a second nickel-charged affinity capture column.Samples from the purification procedure were assessed by SDS-PAGE. Thefinal purified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

Referring to FIG. 12, using the methodology outlined in Example 13, aLHN/C-RGD fusion protein was purified from E. coli BL21 cells. Briefly,the soluble products obtained following cell disruption were applied toa nickel-charged affinity capture column. Bound proteins were elutedwith 100 mM imidazole, treated with Factor Xa to activate the fusionprotein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

Referring to FIG. 13, using the methodology outlined in Example 14, aLHN/C-cyclic RGD fusion protein was purified from E. coli BL21 cells.Briefly, the soluble products obtained following cell disruption wereapplied to a nickel-charged affinity capture column. Bound proteins wereeluted with 100 mM imidazole, treated with Factor Xa to activate thefusion protein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

Referring to FIG. 14, using the methodology outlined in Example 15, aLC/C-RGD-HN/C fusion protein was purified from E. coli BL21 cells.Briefly, the soluble products obtained following cell disruption wereapplied to a nickel-charged affinity capture column. Bound proteins wereeluted with 100 mM imidazole, treated with Factor Xa to activate thefusion protein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

Referring to FIG. 15, using the methodology outlined in example 16,BoNT/B (●), LHN/B (▪) and LHN/B-EGF (▴) were assayed for VAMP cleavageactivity.

Referring to FIG. 16, using the methodology outlined in Example 17, theactivity of EGF-LHN/C(SXN100501) and EGF-LHN/B (SXN100328) was assessedin THP-1 immune cells. The quantity of secreted IL-8 was determined byLuminex-based technology. Data are presented as % of LPS control.

Referring to FIG. 17, using the methodology outlined in Example 18, theactivity of EGF-LHN/C (SXN100501) and EGF-LHN/B (SXN100328) was assessedin RPMI-8226 immune cells. The quantity of secreted IL-10 was determinedby Luminex-based technology. Data are presented as % of LPS control.

Referring to FIG. 18, using the methodology outlined in Example 19, theactivity of EGF-LHN/C (SXN100501) and EGF-LHN/B (SXN100328) andCP-RGD-LHN/C (SXN100221) was assessed in PBMC immune cells. The quantityof secreted IL-8 was determined by Luminex-based technology. Data arepresented as % of LPS control.

Referring to FIG. 19, using the methodology outlined in Example 20, theactivity of EGF-LHN/C (SXN100501) and EGF-LHN/B (SXN100328) andCP-RGD-LHN/C (SXN100221) was assessed in PBMC immune cells. The quantityof secreted IP-10 was determined by Luminex-based technology. Data arepresented as % of PHA control.

EXAMPLES Example 1 Production of a Conjugate of a Lectin from Triticumvulgaris and LH_(N)/A

Materials

Lectin from Triticum vulgaris (Wheat Germ Agglutinin-WGA) was obtainedfrom Sigma Ltd.

SPDP was from Pierce Chemical Co.

PD-10 desalting columns were from Pharmacia.

Dimethylsulphoxide (DMSO) was kept anhydrous by storage over a molecularsieve.

Denaturing sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE) and non-denaturing polyacrylamide gel electrophoresis wasperformed using gels and reagents from Novex.

Additional reagents were obtained from Sigma Ltd.

LH_(N)/A was prepared according to a previous method (Shone, C. C. andTranter, H. S. (1995) in “Clostridial Neurotoxins—The molecularpathogenesis of tetanus and botulism”, (Montecucco, C., Ed.), pp.152-160, Springer). FPLC chromatography media and columns were obtainedfrom Amersham Pharmacia Biotech, UK. Affi-gel® Hz matrix and materialswere from BioRad, UK.

Preparation of an Anti-BoNT/A Antibody-Affinity Column

An antibody-affinity column was prepared with specific monoclonalantibodies essentially as suggested by the manufacturers.quadrature.protocol. Briefly, monoclonal antibodies 5BA2.3 & 5BA9.3 which havedifferent epitope recognition in the H_(C) domain (Hallis, B., Fooks,S., Shone, C. and Hambleton, P. (1993) in “Botulinum and TetanusNeurotoxins”, (DasGupta, B. R., Ed.), pp. 433-436, Plenum Press, NewYork) were purified from mouse hybridoma tissue culture supernatant byProtein G (Amersham Pharmacia Biotech) chromatography. These antibodiesrepresent a source of BoNT/A H_(C)-specific binding molecules and can beimmobilised to a matrix or used free in solution to bind BoNT/A. In thepresence of partially purified LH_(N)/A (which has no H_(C) domain)these antibodies will only bind to BoNT/A. The antibodies 5BA2.3 &5BA9.3 were pooled in a 3:1 ratio and two mg of the pooled antibody wasoxidised by the addition of sodium periodate (final concentration of0.2%) prior coupling to 1 ml Affi-Gel Hz™ gel (16 hours at roomtemperature). Coupling efficiencies were routinely greater than 65%. Thematrix was stored at 4° C. in the presence of 0.02% sodium azide.

Purification Strategy for the Preparation of Pure LH_(N)/A

BoNT/A was treated with 17 .mu.g trypsin per mg BoNT/A for a period of72-120 hours. After this time no material of 150 kDa was observed bySDS-PAGE and Coomassie blue staining. The trypsin digested sample waschromatographed (FPLC system, Amersham Pharmacia Biotech) on a Mono Qcolumn (HR5/5) to remove trypsin and separate the majority of BoNT/Afrom LH_(N)/A. The crude sample was loaded onto the column at pH 7 in 20mM HEPES, 50 mM NaCl and 2 ml LH_(N)/A fractions eluted in a NaClgradient from 50 mM to 150 mM. The slightly greater pl of BoNT/A (6.3)relative to LH_(N)/A (5.2) encouraged any BoNT/A remaining aftertrypsinisation to elute from the anion exchange column at a lower saltconcentration than LH_(N)/A. LH_(N)/A containing fractions (asidentified by SDS-PAGE) were pooled for application to the antibodycolumn.

The semi-purified LH_(N)/A mixture was applied and reapplied at least 3times to a 1-2 ml immobilised monoclonal antibody matrix at 20° C. Aftera total of 3 hours in contact with the immobilised antibodies, theLH_(N)/A-enriched supernatant was removed. Entrapment of the BoNT/Acontaminant, rather than specifically binding the LH_(N)/A, enables theelution conditions to be maintained at the optimum for LH_(N) stability.The use of harsh elution conditions e.g. low pH, high salt, chaotropicions, which may have detrimental effects on LH_(N) polypeptide foldingand enzymatic activity, are therefore avoided. Treatment of theimmobilised antibody column with 0.2M glycine/HCl pH2.5 resulted inregeneration of the column and elution of BoNT/A-reactive proteins of150 kDa.

The LH_(N)/A enriched sample was then applied 2 times to a 1 ml HiTrap™Protein G column (Amersham Pharmacia Biotech) at 20° C. Protein G wasselected since it has a high affinity for mouse monoclonal antibodies.This step was included to remove BoNT/A-antibody complexes that mayleach from the immunocolumn. Antibody species bind to the Protein Gmatrix allowing purified LH_(N)/A to elute, essentially by the method ofShone C. C., Hambleton, P., and Melling, J. 1987, Eur. J. Biochem. 167,175-180, and as described in PCT/GB00/03519.

Methods

The lyophilised lectin was rehydrated in phosphate buffered saline (PBS)to a final concentration of 10 mg/ml. Aliquots of this solution werestored at −20° C. until use.

The WGA was reacted with an equal concentration of SPDP by the additionof a 10 mM stock solution of SPDP in DMSO with mixing. After one hour atroom temperature the reaction was terminated by desalting into PBS overa PD-10 column.

The thiopyridone leaving group was removed from the product to release afree —SH group by reduction with dithiothreitol (DTT; 5 mM; 30 min). Thethiopyridone and DTT were removed by once again desalting into PBS overa PD-10 column.

The LH_(N)/A was desalted into PBSE (PBS containing 1 mM EDTA). Theresulting solution (0.5-1.0 mg/ml) was reacted with a four-fold molarexcess of SPDP by addition of a 10 mM stock solution of SPDP in DMSO.After 3 h at room temperature the reaction was terminated by desaltingover a PD-10 column into PBSE.

A portion of the derivatized LH_(N)/A was removed from the solution andreduced with DTT (5 mM, 30 min). This sample was analyzedspectrophotometrically at 280 nm and 343 nm to determine the degree ofderivatisation. The degree of derivatisation achieved was 3.53+/−0.59mol/mol.

The bulk of the derivatized LH_(N)/A and the derivatized WGA were mixedin proportions such that the WGA was in greater than three-fold molarexcess. The conjugation reaction was allowed to proceed for >16 h at 4°C.

The product mixture was centrifuged to clear any precipitate that haddeveloped. The supernatant was concentrated by centrifugation throughconcentrators (with 10000 molecular weight exclusion limit) beforeapplication to a Superose 12 column on an FPLC chromatography system(Pharmacia). The column was eluted with PBS and the elution profilefollowed at 280 nm.

Fractions were analyzed by SDS-PAGE on 4-20% polyacrylamide gradientgels, followed by staining with Coomassie Blue. The major conjugateproducts have an apparent molecular mass of between 106-150 kDa, theseare separated from the bulk of the remaining unconjugated LH_(N)/A andmore completely from the unconjugated WGA. Fractions containingconjugate were pooled prior to addition to PBS-washedN-acetylglucosamine-agarose. Lectin-containing proteins (i.e.WGA-LH_(N)/A conjugate) remained bound to the agarose during washingwith PBS to remove contaminants (predominantly unconjugated LH_(N)/A).WGA-LH_(N)/A conjugate was eluted from the column by the addition of0.3M N-acetylglucosamine (in PBS) and the elution profile followed at280 nm. See FIG. 1 for SDS-PAGE profile of the whole purificationscheme.

The fractions containing conjugate were pooled, dialysed against PBS,and stored at 4° C. until use.

Example 2 Activity of WGA-LH_(N)/A in Cultured Endocrine Cells (HIT-T15)

The hamster pancreatic B cell line HIT-T15 is an example of a cell lineof endocrine origin. It thus represents a model cell line for theinvestigation of inhibition of release effects of the agents. HIT-T15cells possess surface moieties that allow for the binding, andinternalisation, of WGA-LH_(N)/A.

In contrast, HIT-T15 cells lack suitable receptors for clostridialneurotoxins and are therefore not susceptible to botulinum neurotoxins(BoNTs).

FIG. 2 illustrates the inhibition of release of insulin from HIT-T15cells after prior incubation with WGA-LH_(N)/A. It is clear thatdose-dependent inhibition is observed, indicating that WGA-LH_(N)/A caninhibit the release of insulin from an endocrine cell model.

Inhibition of insulin release was demonstrated to correlate withcleavage of the SNARE protein, SNAP-25 (FIG. 3). Thus, inhibition ofrelease of chemical messenger is due to a clostridialendopeptidase-mediated effects of SNARE-protein cleavage.

Materials

Insulin radioimmunoassay kits were obtained from Linco Research Inc.,USA. Western blotting reagents were obtained from Novex.

Methods

HIT-T15 cells were seeded onto 12 well plates and cultured in RPMI-1640medium containing 5% foetal bovine serum, 2 mM L-glutamine for 5 daysprior to use. WGA-LH_(N)/A was applied for 4 hours on ice, the cellswere washed to remove unbound WGA-LH_(N)/A, and the release of insulinassayed 16 hours later. The release of insulin from HIT-T15 cells wasassessed by radioimmunoassay exactly as indicated by the manufacturer'sinstructions.

Cells were lysed in 2M acetic acid/0.1% TFA. Lysates were dried thenresuspended in 0.1 M Hepes, pH 7.0. To extract the membrane proteinsTriton-X-114 (10%, v/v) was added and incubated at 4° C. for 60 min. Theinsoluble material was removed by centrifugation and the supernatantswere warmed to 37° C. for 30 min. The resulting two phases wereseparated by centrifugation and the upper phase discarded. The proteinsin the lower phase were precipitated with chloroform/methanol foranalysis by Western blotting.

The samples were separated by SDS-PAGE and transferred tonitrocellulose. Proteolysis of SNAP-25, a crucial component of theneurosecretory process and the substrate for the zinc-dependentendopeptidase activity of BoNT/A, was then detected by probing with anantibody (SMI-81) that recognises both the intact and cleaved forms ofSNAP-25.

Example 3 Activity of WGA-LH_(N)/A in Cultured Neuroendocrine Cells(PC12)

The rat pheochromocytoma PC12 cell line is an example of a cell line ofneuroendocrine origin. In its undifferentiated form it has propertiesassociated with the adrenal chromaffin cell [Greene and Tischler, in“Advances in Cellular Neurobiology” (Federoff and Hertz, eds), Vol. 3, p373-414. Academic Press, New York, [982]. It thus represents a modelcell line for the investigation of inhibition of release effects of theagents. PC12 cells possess surface moieties that allow for the binding,and internalisation, of WGA-LH_(N)/A. FIG. 4 illustrates the inhibitionof release of noradrenaline from PC12 cells after prior incubation withWGA-LH_(N)/A. It is clear that dose-dependent inhibition is observed,indicating that WGA-LH_(N)/A can inhibit the release of hormone from aneuroendocrine cell model. Comparison of the inhibition effects observedwith conjugate and the untargeted LH_(N)/A demonstrate the requirementfor a targeting moiety (TM) for efficient inhibition of transmitterrelease.

Methods

PC12 cells were cultured on 24 well plates in RPMI-1640 mediumcontaining 10% horse serum, 5% foetal bovine serum, 1% L-glutamine.Cells were treated with a range of concentrations of WGA-LH_(N)/A forthree days. Secretion of noradrenaline was measured by labelling cellswith [³\H]-noradrenaline (2 μCi/ml, 0.5 ml/well) for 60 min. Cells werewashed every 15 min for 1 hour then basal release determined byincubation with a balanced salt solution containing 5 mM KCl for 5 min.Secretion was stimulated by elevating the concentration of extracellularpotassium (100 mM KCl) for 5 min. Radioactivity in basal and stimulatedsuperfusates was determined by scintillation counting. Secretion wasexpressed as a percentage of the total uptake and stimulated secretionwas calculated by subtracting basal. Inhibition of secretion wasdose-dependent with an observed IC₅₀ of 0.63+/−0.15 μg/ml (n=3).Inhibition was significantly more potent when compared to untargetedendopeptidase (LH_(N)/A in FIG. 4). Thus WGA-LH_(N)/A inhibits releaseof neurotransmitter from a model neuroendocrine cell type.

Example 4 Expression and Purification of Catalytically ActiveRecombinant LH_(N)/B

The coding region for LH_(N)/B was inserted in-frame to the 3′ of thegene encoding maltose binding protein (MBP) in the expression vectorpMAL (New England Biolabs). In this construct, the expressed MBP andLH_(N)/B polypeptides are separated by a Factor Xa cleavage site.

Expression of the MBP-LH_(N)/B in E. coli TG1 was induced by addition ofIPTG to the growing culture at an approximate OD600 nm of 0.8.Expression was maintained for a further 3 hours in the presence ofinducing agent prior to harvest by centrifugation. The recovered cellpaste was stored at −20° C. until required.

The cell paste was resuspended in resuspension buffer (50 mM HepespH7.5+150 mM NaCl⁺ a variety of protease inhibitors) at 6 ml buffer pergram paste. To this suspension was added lysozyme to a finalconcentration of 1 mg/ml. After 10 min at 0° C., the suspension wassonicated for 6×30 seconds at 24μ at 0° C. The broken cell paste wasthen centrifuged to remove cell debris and the supernatant recovered forchromatography.

In some situations, the cell paste was disrupted by using proprietarydisruption agents such as BugBuster™ (Novagen) as per the manufacturersprotocol. These agents were satisfactory for disruption of the cells toprovide supernatant material for affinity chromatography.

The supernatant was applied to an immobilised amylose matrix at 0.4ml/min to facilitate binding of the fusion protein. After binding, thecolumn was washed extensively with resuspension buffer to removecontaminating proteins. Bound proteins were eluted by the addition ofelution buffer (resuspension buffer+10 mM maltose) and fractionscollected. Eluted fractions containing protein were pooled for treatmentwith Factor Xa.

On some occasions a further purification step was incorporated into thescheme, prior to the addition of Factor Xa. In these instances, theeluted fractions were made to 5 mM DTT and applied to a Pharmacia Mono-QHR5/5 column (equilibrated in resuspension buffer) as part of an FPLCsystem. Proteins were bound to the column at 150 mM NaCl, beforeincreased to 500 mM NaCl over a gradient. Fractions were collected andanalysed for the presence of MBP-LH_(N)/B by Western blotting (probeantibody=guinea pig anti-BoNT/B or commercially obtained anti-MBP).

Cleavage of the fusion protein by Factor Xa was as described in theprotocol supplied by the manufacturer (New England Biolabs). Cleavage ofthe fusion protein resulted in removal of the MBP fusion tag andseparation of the LC and H_(N) domains of LH_(N)/B. Passage of thecleaved mixture through a second immobilised maltose column removed freeMBP from the mixture to leave purified disulphide-linked LH_(N)/B. Thismaterial was used for conjugation.

See FIG. 5 for an illustration of the purification of LH_(N)/B.

See FIG. 6 for an illustration of the in vitro catalytic activity ofLH_(N)/B.

Example 5 Production of a Conjugate of a Lectin from Triticum vulgarisand LH_(N)/B

Materials

Lectin from Triticum vulgaris (WGA) was obtained from Sigma Ltd.

LH_(N)/B was prepared as described in Example 4.

SPDP was from Pierce Chemical Co.

PD-10 desalting columns were from Pharmacia.

Dimethylsulphoxide (DMSO) was kept anhydrous by storage over a molecularsieve.

Polyacrylamide gel electrophoresis was performed using gels and reagentsfrom Novex.

Additional reagents were obtained from Sigma Ltd.

Methods

The lyophilised lectin was rehydrated in phosphate buffered saline (PBS)to a final concentration of 10 mg/ml. Aliquots of this solution werestored at −20° C. until use.

The WGA was reacted with an equal concentration of SPDP by the additionof a 10 mM stock solution of SPDP in DMSO with mixing. After one hour atroom temperature the reaction was terminated by desalting into PBS overa PD-10 column.

The thiopyridone leaving group was removed from the product to release afree —SH group by reduction with dithiothreitol (DTT; 5 mM; 30 min). Thethiopyridone and DTT were removed by once again desalting into PBS overa PD-10 column.

The recLH_(N)/B was desalted into PBS. The resulting solution (0.5-1.0mg/ml) was reacted with a four-fold molar excess of SPDP by addition ofa 10 mM stock solution of SPDP in DMSO. After 3 h at room temperaturethe reaction was terminated by desalting over a PD-10 column into PBS.

A portion of the derivatized recLH_(N)/B was removed from the solutionand reduced with DTT (5 mM, 30 min). This sample was analysedspectrophotometrically at 280 nm and 343 nm to determine the degree ofderivatisation.

The bulk of the derivatized recLH_(N)/B and the derivatized WGA weremixed in proportions such that the WGA was in greater than three-foldmolar excess. The conjugation reaction was allowed to proceed for >16 hat 4° C.

The product mixture was centrifuged to clear any precipitate that haddeveloped. The supernatant was concentrated by centrifugation throughconcentrators (with 10000 molecular weight exclusion limit) beforeapplication to a Superdex G-200 column on an FPLC chromatography system(Pharmacia). The column was eluted with PBS and the elution profilefollowed at 280 nm.

Fractions were analysed by SDS-PAGE on 4-20% polyacrylamide gradientgels, followed by staining with Coomassie Blue. The major conjugateproducts have an apparent molecular mass of between 106-150 kDa, theseare separated from the bulk of the remaining unconjugated recLH_(N)/Band more completely from the unconjugated WGA. Fractions containingconjugate were pooled prior to addition to PBS-washedN-acetylglucosamine-agarose. Lectin-containing proteins (i.e.WGA-recLH_(N)/B conjugate) remained bound to the agarose during washingwith PBS to remove contaminants (predominantly unconjugatedrecLH_(N)/B). WGA-recLH_(N)/B conjugate was eluted from the column bythe addition of 0.3M N-acetylglucosamine (in PBS) and the elutionprofile followed at 280 nm.

The fractions containing conjugate were pooled, dialysed against PBS,and stored at 4° C. until use.

Example 6 Activity of BoNT/B in Vascular Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) secrete von WillebrandsFactor (vWF) when stimulated with a variety of cell surface receptoragonists including histamine. These cells maintain this property whenprepared from full term umbilical cords and grown in culture (Loesberget al 1983, Biochim. Biophys. Acta. 763, 160-168). The release of vWF byHUVEC thus represents a secretory activity of a non-neuronal cell typederived from the cardiovascular system. FIG. 7 illustrates theinhibition of the histamine stimulated release of vWF by HUVEC whenpreviously treated with BoNT/B in low pH medium. Treatment of cells withtoxins in low pH can be used as a technique for facilitating toxinpenetration of the plasmalemma of cells refractory to exogenouslyapplied clostridial neurotoxins.

This result clearly shows the ability of botulinum neurotoxins toinhibit secretory activity of non-neuronal cells in the cardiovascularsystem (see FIG. 7).

Methods

HUVEC were prepared by the method of Jaffe et al 1973, J. Clin. Invest.52, 2745-2756. Cells were passaged once onto 24 well plates in medium199 supplemented with 10% foetal calf serum, 10% newborn calf serum, 5mM L-glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, 20μg/ml endothelial cell growth factor (Sigma). Cells were treated withDMEM pH 7.4, DMEM pH 4.7 (pH lowered with HCl) or DMEM, pH 4.7 with 500nM BoNT/B for 2.5 hours then washed three times with HUVEC medium. 24hours later cells were washed with a balanced salt solution, pH 7.4 andexposed to this solution for 30 minutes for the establishment of basalrelease. This was removed and BSS containing 1 mM histamine applied fora further 30 minutes. Superfusates were centrifuged to remove anydetached cells and the quantity of vWF determined using an ELISA assayas described by Paleolog et al 1990, Blood. 75, 688-695. Stimulatedsecretion was then calculated by subtracting basal from the histaminestimulated release. Inhibition by BoNT/B treatment at pH 4.7 wascalculated at 27.4% when compared to pH 4.7 treatment alone.

Example 7 Activity of BoNT/B in Mucus Secreting Cells

The LS180 colon carcinoma cell line is recognised as a model of mucinsecreting cells (McCool, D. J., Forstner, J. F. and Forstner, G. G. 1994Biochem. J. 302, 111-118). These cells have been shown to adopt gobletcell morphology and release high molecular weight mucin when stimulatedwith muscarinic agonists (eg carbachol), phorbol esters (PMA) and Ca²⁺ionophores (eg A23187) (McCool, D. J., Forstner, J. F. and Forstner, G.G. 1995 Biochem. J. 312, 125-133). These cells thus represent anon-neuronal cell type derived from the colon which can undergoregulated mucin secretion. FIG. 8 illustrates the inhibition of theionomycin stimulated release of high molecular weight, [³H]-glucosaminelabelled material from LS180 cells by pretreatment with BoNT/B in low pHmedium. Ionomycin is a Ca²⁺ ionophore and treatment of cells with low pHmedium has been previously shown to facilitate toxin entry into cells.

This result clearly shows the ability of botulinum neurotoxins toinhibit secretory activity of non-neuronal cells able to release mucinwhen stimulated with a secretagogue (see FIG. 8).

Methods

Mucin synthesising colon carcinoma LS180 cells were grown on Matrigelcoated 24 well plates in minimum essential medium supplemented with 10%foetal calf serum, 2 mM L-glutamine and 1% non-essential amino acids(Sigma) Cells were treated with pH 7.4 medium, pH 4.7 medium and pH 4.7medium containing 500 nM botulinum neurotoxin type B (BoNT/B) for fourhours then labelled with [³H]-glucosamine (1 μCi/ml, 0.5 ml/well) for 18hours in L15 glucose free medium. Cells were then washed twice with abalanced salt solution (BSS) pH 7.4 and then 0.5 ml of BSS was appliedfor 30 minutes. This material was removed and 0.5 ml of BSS containing10 μM ionomycin applied to stimulate mucin release. The stimulatingsolution was removed and all superfusates centrifuged to remove anydetached cells. Supernatants were then centrifuged at 100,000×g for 1hour. Supernatants were applied to Centricon centrifugal concentratorswith a molecular weight cut-off of 100 kDa and centrifuged (2,500×g)until all liquid had passed through the membrane. Membranes were washedwith BSS by centrifugation three times and then the membranescintillation counted for retained, [³H]-glucosamine labelled highmolecular weight material.

Example 8 Activity of BoNT/B in Inflammatory Cells

The promyelocytic cell line HL60 can be differentiated into neutrophillike cells by the addition of dibutyryl cyclic AMP to the culturemedium. Upon differentiation these cells increase their expression ofcharacteristic enzymes such as .beta.-glucuronidase. In this conditionthese cells therefore represent a model of a phagocytic cell type whichcontributes to the inflammatory response of certain disease states (egrheumatoid arthritis). FIG. 9 illustrates the significant (p>0.05)inhibition of stimulated release of .beta.-glucuronidase from dbcAMPdifferentiated HL60 cells by pre-treatment with BoNT/B in low pH medium.

This result clearly shows the ability of botulinum neurotoxins toinhibit the secretory activity of a non-neuronal cell type which is amodel of the neutrophil a cell which participates in inflammation.

Methods

HL60 cells were cultured in RPMI 1640 medium containing 10% foetal calfserum and 2 mM glutamine. Cells were exposed to low pH and toxin for 2.5hours then washed 3 times and differentiated by the addition ofdibutyryl cyclic AMP (dbcAMP) to a final concentration of 300 μM. Cellswere differentiated for 40 hours and then stimulated release ofβ-glucuronidase activity was determined. Cells were treated withcytochalasin B (5 .mu.M) 5 minutes before stimulation. Cells werestimulated with 1 .mu.M N-formyl-Met-Leu-Phe with 100 μM ATP for 10minutes then centrifuged and the supernatant taken for assay ofβ-glucuronidase activity. Activity was measured in cell lysates and theamount released expressed as a percentage of the total cellular contentof enzyme.

β-glucuronidase activity was determined according to the method ofAbsolom D. R. 1986, (Methods in Enzymology, 132, 160) usingp-Nitrophenyl-β-D-glucuronide as the substrate.

Example 9 Preparation of a LHN/B Backbone Construct

The following procedure creates a clone for use as an expressionbackbone for multidomain fusion expression. This example is based onpreparation of a serotype B based clone (SEQ ID NO:1).

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due tothe lack of restriction sequences within the vector and adjacentsequencing primer sites for easy construct confirmation. The expressionvector is based on the pMAL (NEB) expression vector which has thedesired restriction sequences within the multiple cloning site in thecorrect orientation for construct insertion (BamHI-SalI-PstI-HindIII). Afragment of the expression vector has been removed to create anon-mobilisable plasmid and a variety of different fusion tags have beeninserted to increase purification options.

Preparation of LC/B

The LC/B is created by one of two ways:

The DNA sequence is designed by back translation of the LC/B amino acidsequence (obtained from freely available database sources such asGenBank (accession number P10844) or Swissprot (accession locusBXB_CLOBO) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)). BamHI/SalI recognitionsequences are incorporated at the 5′ and 3′ ends respectively of thesequence maintaining the correct reading frame. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required by thecloning system are removed manually from the proposed coding sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, Sep. 13, 2004). This optimised DNA sequencecontaining the LC/B open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with BamHI and SalI restriction enzyme sequences incorporatedinto the 5′ and 3′ PCR primers respectively. Complementaryoligonucleotide primers are chemically synthesised by a Supplier (forexample MWG or Sigma-Genosys) so that each pair has the ability tohybridize to the opposite strands (3′ ends pointing “towards” eachother) flanking the stretch of Clostridium target DNA, oneoligonucleotide for each of the two DNA strands. To generate a PCRproduct the pair of short oligonucleotide primers specific for theClostridium DNA sequence are mixed with the Clostridium DNA template andother reaction components and placed in a machine (the ‘PCR machine’)that can change the incubation temperature of the reaction tubeautomatically, cycling between approximately 94° C. (for denaturation),55° C. (for oligonucleotide annealing), and 72° C. (for synthesis).Other reagents required for amplification of a PCR product include a DNApolymerase (such as Taq or Pfu polymerase), each of the four nucleotidedNTP building blocks of DNA in equimolar amounts (50-200 μM) and abuffer appropriate for the enzyme optimised for Mg2+ concentration(0.5-5 mM).

The amplification product is cloned into pCR 4 using either, TOPO TAcloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCRproducts (both kits commercially available from Invitrogen). Theresultant clone is checked by sequencing. Any additional restrictionsequences which are not compatible with the cloning system are thenremoved using site directed mutagenesis (for example using Quickchange(Stratagene Inc.)).

Preparation of HN/B Insert

The HN is created by one of two ways:

The DNA sequence is designed by back translation of the HN/B amino acidsequence (obtained from freely available database sources such asGenBank (accession number P10844) or Swissprot (accession locusBXB_CLOBO)) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBack translation tool v2.0 (Entelechon)). A PstI restriction sequenceadded to the N-terminus and XbaI-stop codon-HindIII to the C-terminusensuring the correct reading frame in maintained. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anysequences that are found to be common to those required by the cloningsystem are removed manually from the proposed coding sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage isassessed by reference to software programs such as Graphical Codon UsageAnalyser (Geneart), and the % GC content and codon usage ratio assessedby reference to published codon usage tables (for example GenBankRelease 143, Sep. 13, 2004). This optimised DNA sequence is thencommercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with PstI and XbaI-stop codon-HindIII restriction enzymesequences incorporated into the 5′ and 3′ PCR primers respectively. ThePCR amplification is performed as described above. The PCR product isinserted into pCR 4 vector and checked by sequencing. Any additionalrestriction sequences which are not compatible with the cloning systemare then removed using site directed mutagenesis (for example usingQuickchange (Stratagene Inc.)).

Preparation of the Spacer (LC-HN Linker)

The LC-HN linker can be designed from first principle, using theexisting sequence information for the linker as the template. Forexample, the serotype B linker (in this case defined as the inter-domainpolypeptide region that exists between the cysteines of the disulphidebridge between LC and HN) has the sequence KSVKAPG (SEQ ID NO:30). Thissequence information is freely available from available database sourcessuch as GenBank (accession number P10844) or Swissprot (accession locusBXB_CLOBO). For generation of a specific protease cleavage site, therecognition sequence for enterokinase is inserted into the activationloop to generate the sequence VDEEKLYDDDDKDRWGSSLQ (SEQ ID NO:31). Usingone of a variety of reverse translation software tools (for exampleEditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)), the DNA sequence encoding thelinker region is determined. BamHI/SalI and PstI/XbaI/stop codon/HindIIIrestriction enzyme sequences are incorporated at either end, in thecorrect reading frames. The DNA sequence is screened (using softwaresuch as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequencesincorporated during the back translation. Any sequences that are foundto be common to those required by the cloning system are removedmanually from the proposed coding sequence ensuring common E. coli codonusage is maintained. E. coli codon usage is assessed by reference tosoftware programs such as Graphical Codon Usage Analyser (Geneart), andthe % GC content and codon usage ratio assessed by reference topublished codon usage tables (for example GenBank Release 143, Sep. 13,2004). This optimised DNA sequence is then commercially synthesized (forexample by Entelechon, Geneart or Sigma-Genosys) and is provided in thepCR 4 vector. If it is desired to clone the linker out of pCR 4 vector,the vector (encoding the linker) is cleaved with either BamHI+SalI orPstI+XbaI combination restriction enzymes. This cleaved vector thenserves as the recipient vector for insertion and ligation of either theLC DNA (cleaved with BamHI/SalI) or HN DNA (cleaved with PstI/XbaI).Once the LC or the HN encoding DNA is inserted upstream or downstream ofthe linker DNA, the entire LC-linker or linker-HN DNA fragment can thebe isolated and transferred to the backbone clone.

As an alternative to independent gene synthesis of the linker, thelinker-encoding DNA can be included during the synthesis or PCRamplification of either the LC or HN.

Assembly and Confirmation of the Backbone Clone

The LC or the LC-linker is cut out from the pCR 4 cloning vector usingBamHI/SalI or BamHI/PstI restriction enzymes digests. The pMALexpression vector is digested with the same enzymes but is also treatedwith calf intestinal protease (CIP) as an extra precaution to preventre-circularisation. Both the LC or LC-linker region and the pMAL vectorbackbone are gel purified. The purified insert and vector backbone areligated together using T4 DNA ligase and the product is transformed withTOP10 cells which are then screened for LC insertion using BamHI/SalI orBamHI/PstI restriction digestion. The process is then repeated for theHN or linker-HN insertion into the PstI/HindIII or SalI/HindIIIsequences of the pMAL-LC construct.

Screening with restriction enzymes is sufficient to ensure the finalbackbone is correct as all components are already sequenced confirmed,either during synthesis or following PCR amplification. However, duringthe sub-cloning of some components into the backbone, where similar sizefragments are being removed and inserted, sequencing of a small regionto confirm correct insertion is required.

Example 10 Preparation of a LHN/C Backbone Construct

The following procedure creates a clone for use as an expressionbackbone for multidomain fusion expression. This example is based onpreparation of a serotype C based clone (SEQ ID NO:2).

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due tothe lack of restriction sequences within the vector and adjacentsequencing primer sites for easy construct confirmation. The expressionvector is based on the pMAL (NEB) expression vector which has thedesired restriction sequences within the multiple cloning site in thecorrect orientation for construct insertion (BamHI-SalI-PstI-HindIII). Afragment of the expression vector has been removed to create anon-mobilisable plasmid and a variety of different fusion tags have beeninserted to increase purification options.

Preparation of LC/C

The LC/C is created by one of two ways:

The DNA sequence is designed by back translation of the LC/C amino acidsequence (obtained from freely available database sources such asGenBank (accession number P18640) or Swissprot (accession locusBXC1_CLOBO) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)). BamHI/SalI recognitionsequences are incorporated at the 5′ and 3′ ends respectively of thesequence maintaining the correct reading frame. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required by thecloning system are removed manually from the proposed coding sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, Sep. 13, 2004). This optimised DNA sequencecontaining the LC/C open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with BamHI and SalI restriction enzyme sequences incorporatedinto the 5′ and 3′ PCR primers respectively. Complementaryoligonucleotide primers are chemically synthesised by a Supplier (forexample MWG or Sigma-Genosys) so that each pair has the ability tohybridize to the opposite strands (3′ ends pointing “towards” eachother) flanking the stretch of Clostridium target DNA, oneoligonucleotide for each of the two DNA strands. To generate a PCRproduct the pair of short oligonucleotide primers specific for theClostridium DNA sequence are mixed with the Clostridium DNA template andother reaction components and placed in a machine (the ‘PCR machine’)that can change the incubation temperature of the reaction tubeautomatically, cycling between approximately 94° C. (for denaturation),55° C. (for oligonucleotide annealing), and 72° C. (for synthesis).Other reagents required for amplification of a PCR product include a DNApolymerase (such as Taq or Pfu polymerase), each of the four nucleotidedNTP building blocks of DNA in equimolar amounts (50-200 μM) and abuffer appropriate for the enzyme optimised for Mg2+ concentration(0.5-5 mM).

The amplification product is cloned into pCR 4 using either, TOPO TAcloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCRproducts (both kits commercially available from Invitrogen). Theresultant clone is checked by sequencing. Any additional restrictionsequences which are not compatible with the cloning system are thenremoved using site directed mutagenesis (for example using Quickchange(Stratagene Inc.)).

Preparation of HN/C Insert

The HN is created by one of two ways:

The DNA sequence is designed by back translation of the HN/C amino acidsequence (obtained from freely available database sources such asGenBank (accession number P18640) or Swissprot (accession locusBXC1_CLOBO)) using one of a variety of reverse translation softwaretools (for example EditSeq best E. coli reverse translation (DNASTARInc.), or Back translation tool v2.0 (Entelechon)). A PstI restrictionsequence added to the N-terminus and XbaI-stop codon-HindIII to theC-terminus ensuring the correct reading frame in maintained. The DNAsequence is screened (using software such as MapDraw, DNASTAR Inc.) forrestriction enzyme cleavage sequences incorporated during the backtranslation. Any sequences that are found to be common to those requiredby the cloning system are removed manually from the proposed codingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, Sep. 13, 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with PstI and XbaI-stop codon-HindIII restriction enzymesequences incorporated into the 5′ and 3′ PCR primers respectively. ThePCR amplification is performed as described above. The PCR product isinserted into pCR 4 vector and checked by sequencing. Any additionalrestriction sequences which are not compatible with the cloning systemare then removed using site directed mutagenesis (for example usingQuickchange (Stratagene Inc.)).

Preparation of the Spacer (LC-HN Linker)

The LC-HN linker can be designed from first principle, using theexisting sequence information for the linker as the template. Forexample, the serotype C linker (in this case defined as the inter-domainpolypeptide region that exists between the cysteines of the disulphidebridge between LC and HN) has the sequence HKAIDGRSLYNKTLD (SEQ IDNO:32). This sequence information is freely available from availabledatabase sources such as GenBank (accession number P18640) or Swissprot(accession locus BXC1_CLOBO). For generation of a specific proteasecleavage site, the recognition sequence for enterokinase is insertedinto the activation loop to generate the sequenceVDGIITSKTKSDDDDKNKALNLQ (SEQ ID NO:33). Using one of a variety ofreverse translation software tools (for example EditSeq best E. colireverse translation (DNASTAR Inc.), or Backtranslation tool v2.0(Entelechon)), the DNA sequence encoding the linker region isdetermined. BamHI/SalI and PstI/XbaI/stop codon/HindIII restrictionenzyme sequences are incorporated at either end, in the correct readingframes. The DNA sequence is screened (using software such as MapDraw,DNASTAR Inc.) for restriction enzyme cleavage sequences incorporatedduring the back translation. Any sequences that are found to be commonto those required by the cloning system are removed manually from theproposed coding sequence ensuring common E. coli codon usage ismaintained. E. coli codon usage is assessed by reference to softwareprograms such as Graphical Codon Usage Analyser (Geneart), and the % GCcontent and codon usage ratio assessed by reference to published codonusage tables (for example GenBank Release 143, Sep. 13, 2004). Thisoptimised DNA sequence is then commercially synthesized (for example byEntelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4vector. If it is desired to clone the linker out of pCR 4 vector, thevector (encoding the linker) is cleaved with either BamHI+SalI orPstI+XbaI combination restriction enzymes. This cleaved vector thenserves as the recipient vector for insertion and ligation of either theLC DNA (cleaved with BamHI/SalI) or HN DNA (cleaved with PstI/XbaI).Once the LC or the HN encoding DNA is inserted upstream or downstream ofthe linker DNA, the entire LC-linker or linker-HN DNA fragment can thebe isolated and transferred to the backbone clone.

As an alternative to independent gene synthesis of the linker, thelinker-encoding DNA can be included during the synthesis or PCRamplification of either the LC or HN.

Assembly and Confirmation of the Backbone Clone

The LC or the LC-linker is cut out from the pCR 4 cloning vector usingBamHI/SalI or BamHI/PstI restriction enzymes digests. The pMALexpression vector is digested with the same enzymes but is also treatedwith calf intestinal protease (CIP) as an extra precaution to preventre-circularisation. Both the LC or LC-linker region and the pMAL vectorbackbone are gel purified. The purified insert and vector backbone areligated together using T4 DNA ligase and the product is transformed withTOP10 cells which are then screened for LC insertion using BamHI/SalI orBamHI/PstI restriction digestion. The process is then repeated for theHN or linker-HN insertion into the PstI/HindIII or SalI/HindIIIsequences of the pMAL-LC construct.

Screening with restriction enzymes is sufficient to ensure the finalbackbone is correct as all components are already sequenced confirmed,either during synthesis or following PCR amplification. However, duringthe sub-cloning of some components into the backbone, where similar sizefragments are being removed and inserted, sequencing of a small regionto confirm correct insertion is required.

Example 11 Construction, Expression, and Purification of a LHN/C-EGFFusion Protein

Preparation of Spacer-EGF Insert

For presentation of an EGF sequence at the C-terminus of the HN domain,a DNA sequence is designed to flank the spacer and targeting moiety (TM)regions allowing incorporation into the backbone clone (SEQ ID NO:2).The DNA sequence can be arranged as BamHI-SalI-PstI-XbaI-spacer-EGF-stopcodon-HindIII (SEQ ID NO:3). The DNA sequence can be designed using oneof a variety of reverse translation software tools (for example EditSeqbest E. coli reverse translation (DNASTAR Inc.), or Backtranslation toolv2.0 (Entelechon)). Once the TM DNA is designed, the additional DNArequired to encode the preferred spacer is created in silico. It isimportant to ensure the correct reading frame is maintained for thespacer, EGF and restriction sequences and that the XbaI sequence is notpreceded by the bases, TC which would result on DAM methylation. The DNAsequence is screened for restriction sequence incorporated and anyadditional sequences are removed manually from the remaining sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is thencommercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

Insertion of Spacer-EGF into Backbone

In order to create a LC-linker-HN-spacer-EGF construct (SEQ ID NO:4)using the backbone construct (SEQ ID NO:2) and the newly synthesised pCR4-spacer-TM vector encoding the EGF TM (SEQ ID NO:3), the followingtwo-step method is employed. Firstly, the HN domain is excised from thebackbone clone using restriction enzymes PstI and XbaI and ligated intosimilarly digested pCR 4-spacer-EGF vector. This creates anHN-spacer-EGF ORF in pCR 4 that can be excised from the vector usingrestriction enzymes PstI and HindIII for subsequent ligation intosimilarly cleaved backbone or expression construct. The final constructcontains the LC-linker-HN-spacer-EGF ORF (SEQ ID NO:4) for transfer intoexpression vectors for expression to result in a fusion protein of thesequence illustrated in SEQ ID NO:5.

Screening with restriction enzymes is sufficient to ensure the finalbackbone is correct as all components are already sequenced confirmed,either during synthesis or following PCR amplification. However, duringthe sub-cloning of some components into the backbone, where similar sizefragments are being removed and inserted, sequencing of a small regionto confirm correct insertion is required.

Alternative Construction Approach

As an alternative to the methodologies described above for theconstruction of LHN/C-EGF, complete gene synthesis has been used tocreate a single DNA insert that encodes the LC, the HN, linkers, spacersand a protease activation site. The synthetic DNA is designed to have aNdeI restriction site at the 5′ end and a HindIII restriction site atthe 3′ end to facilitate direct cloning into expression vectors. Thesequence of the engineered coding region is subject to the same codonutilisation analysis as described above. The sequence of the syntheticDNA is illustrated in SEQ ID NO:19, and the protein that it encodes isillustrated in SEQ ID NO:20.

Expression of LHN/C-EGF Fusion Protein

Expression of the LHN/C-EGF fusion protein is achieved using thefollowing protocol. Inoculate 100 ml of modified TB containing 0.2%glucose and 100 mg/ml ampicillin in a 250 ml flask with a single colonyfrom the LHN/C-EGF expression strain. Grow the culture at 37° C., 225rpm for 16 hours. Inoculate 1 L of modified TB containing 0.2% glucoseand 100 □g/ml ampicillin in a 2 L flask with 10 ml of overnight culture.Grow cultures at 37° C. until an approximate OD600 nm of 0.5 is reachedat which point reduce the temperature to 16° C. After 1 hour induce thecultures with 1 mM IPTG and grow at 16° C. for a further 16 hours.

Purification of LHN/C-EGF Fusion Protein

Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2 200 mM NaCl andapproximately 10 g of E. coli BL21 cell paste. Sonicate the cell pasteon ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22microns ensuring the sample remains cool. Spin the lysed cells at 18 000rpm, 4° C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO4charged Chelating column (20-30 ml column is sufficient) equilibratedwith 50 mM HEPES pH 7.2 200 mM NaCl. Using a step gradient of 10 and 40mM imidazole, wash away the non-specific bound protein and elute thefusion protein with 100 mM imidazole. Dialyse the eluted fusion proteinagainst 5 L of 50 mM HEPES pH 7.2 200 mM NaCl at 4° C. overnight andmeasure the OD of the dialysed fusion protein. Add 1 unit of factor Xaper 100 □g fusion protein and incubate at 25° C. static overnight. Loadonto a 0.1 M NiSO4 charged Chelating column (20-30 ml column issufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCl. Washcolumn to baseline with 50 mM HEPES pH 7.2 200 mM NaCl. Using a stepgradient of 10 and 40 mM imidazole, wash away the non-specific boundprotein and elute the fusion protein with 100 mM imidazole. Dialyse theeluted fusion protein against 5 L of 50 mM HEPES pH 7.2 200 mM NaCl at4° C. overnight and concentrate the fusion to about 2 mg/ml, aliquotsample and freeze at −20° C. Test purified protein using OD, BCA andpurity analysis. FIG. 8 demonstrates the purified protein as analysed beSDS-PAGE.

Example 12 Construction, Expression and Purification of a LHN/B-EGFFusion Protein

The LC-HN linker is designed using the methods described in example 11using the B serotype linker arranged asBamHI-SalI-PstI-XbaI-spacer-EGF-stop codon-HindIII (SEQ ID NO:3). TheLHN/B-EGF fusion is then assembled using the LHN/B backbone clone (SEQID NO:1) made using the methods described in example 9 and constructedusing methods described in example 11. The final construct contains theLC-linker-HN-spacer-EGF ORF (SEQ ID NO:6) for transfer into expressionvectors for expression to result in a fusion protein of the sequenceillustrated in SEQ ID NO:7. The resultant expression plasmid, pMALLHN/B-EGF is transformed into E. coli BL21 for recombinant proteinexpression. Expression and purification of the fusion protein wascarried out as described in example 6 except that enterokinase replacedfactor Xa in the activation of the fusion protein. FIG. 9 demonstratesthe purified protein as analysed by SDS-PAGE.

Example 13 Preparation and Purification of a LHN/C-RGD Fusion Protein

Preparation of Spacer-RGD Insert

For presentation of an RGD sequence at the C-terminus of the HN domain,a DNA sequence is designed to flank the spacer and TM regions allowingincorporation into the backbone clone (SEQ ID NO:2). The DNA sequencecan be arranged as BamHI-SalI-PstI-XbaI-spacer-SpeI-RGD-stopcodon-HindIII (SEQ ID NO:8). The DNA sequence can be designed using oneof a variety of reverse translation software tools (for example EditSeqbest E. coli reverse translation (DNASTAR Inc.), or Backtranslation toolv2.0 (Entelechon)). Once the TM DNA is designed, the additional DNArequired to encode the preferred spacer is created in silico. It isimportant to ensure the correct reading frame is maintained for thespacer, RGD and restriction sequences and that the XbaI sequence is notpreceded by the bases, TC which would result on DAM methylation. The DNAsequence is screened for restriction sequence incorporated and anyadditional sequences are removed manually from the remaining sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is thencommercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

Insertion of Spacer-RGD into Backbone

In order to create a LC-linker-HN-spacer-RGD construct (SEQ ID NO:9)using the backbone construct (SEQ ID NO:2) and the newly synthesised pCR4-spacer-TM vector encoding the RGD TM (SEQ ID NO:8), the followingtwo-step method is employed. Firstly, the HN domain is excised from thebackbone clone using restriction enzymes PstI and XbaI and ligated intosimilarly digested pCR 4-spacer-RGD vector. This creates anHN-spacer-RGD ORF in pCR 4 that can be excised from the vector usingrestriction enzymes PstI and HindIII for subsequent ligation intosimilarly cleaved backbone or expression construct. The final constructcontains the LC-linker-HN-spacer-RGD ORF (SEQ ID NO:9) for transfer intoexpression vectors for expression to result in a fusion protein of thesequence illustrated in SEQ ID NO:10.

Screening with restriction enzymes is sufficient to ensure the finalbackbone is correct as all components are already sequenced confirmed,either during synthesis or following PCR amplification. However, duringthe sub-cloning of some components into the backbone, where similar sizefragments are being removed and inserted, sequencing of a small regionto confirm correct insertion is required.

Expression and purification of the fusion protein was carried out asdescribed in example 11. FIG. 10 demonstrates the purified protein asanalysed by SDS-PAGE.

Example 14 Preparation and Purification of a LHN/C-cyclic RGD FusionProtein

The LC-HN linker can be designed using the methods described in example13 using the C serotype linker arranged asBamHI-SalI-PstI-XbaI-spacer-SpeI-cyclic RGD-stop codon-HindIII (SEQ IDNO:11). The LHN/C-cyclic RGD fusion is then assembled using the LHN/Cbackbone clone (SEQ ID NO:2) made using the methods described in example10 and constructed using methods described in example 13. The finalconstruct contains the LC-linker-HN-spacer-cyclic RGD ORF (SEQ ID NO:11)for transfer into expression vectors for expression to result in afusion protein of the sequence illustrated in SEQ ID NO:13. Theresultant expression plasmid, pMAL LHN/C-cyclic RGD was transformed intoE. coli BL21 for recombinant protein expression. Expression andpurification of the fusion protein was carried out as described inexample 11. FIG. 11 demonstrates the purified protein as analysed bySDS-PAGE.

Example 15 Preparation and Purification of a LC/C-RGD-HN/C FusionProtein

In order to create the LC-linker-RGD-spacer-HN construct (SEQ ID NO:15),the pCR 4 vector encoding the linker (SEQ ID NO:14) is cleaved withBamHI+SalI restriction enzymes. This cleaved vector then serves as therecipient vector for insertion and ligation of the LC/C DNA (SEQ IDNO:2) cleaved with BamHI+SalI. The resulting plasmid DNA is then cleavedwith PstI+XbaI restriction enzymes and serves as the recipient vectorfor the insertion and ligation of the HN/C DNA (SEQ ID NO:2) cleavedwith PstI+XbaI. The final construct contains the LC-linker-RGD-spacer-HNORF (SEQ ID NO:15) for transfer into expression vectors for expressionto result in a fusion protein of the sequence illustrated in SEQ IDNO:16. The resultant expression plasmid, pMAL LC/C-RGD-HN/C wastransformed into E. coli BL21 for recombinant protein expression.Expression and purification of the fusion protein was carried out asdescribed in example 11. FIG. 12 demonstrates the purified protein asanalysed by SDS-PAGE.

Alternative Construction Approach

As an alternative to the methodologies described above for theconstruction of LC-linker-RGD-spacer-HN, complete gene synthesis hasbeen used to create a single DNA insert that encodes the LC, the HN,linkers, spacers and a protease activation site. The synthetic DNA isdesigned to have a NdeI restriction site at the 5′ end and a HindIIIrestriction site at the 3′ end to facilitate direct cloning intoexpression vectors. The sequence of the engineered coding region issubject to the same codon utilisation analysis as described above. Thesequence of the synthetic DNA is illustrated in SEQ ID NO:17, and theprotein that it encodes is illustrated in SEQ ID NO:18.

Example 16 VAMP Cleavage Activity Assay

A range of concentrations of LHN/B-EGF in cleavage buffer (50 mM HEPESpH7.4, 10 mM DTT, 20 □M ZnCl2, 1% FBS) are incubated with biotinylatedVAMP substrate (1 mg/ml) for two hours at 37° C. in a shaking incubator.The cleavage reaction is transferred to a washed 96-well streptavidincoated plate and incubated at 37° C. in a shaking incubator for 5minutes. The plate is washed three times with PBS-0.1% tween-20 (PBS-T).The wells are blocked with blocking buffer (5% FCS in PBS-T) for 1 hourat 37° C. The primary antibody (anti-FESS) is added at a dilution of 1in 500 in blocking buffer and the plate is incubated at 37° C. for 1hour. The plate is washed three times with PBS-T and the secondaryantibody (anti guinea pig HRP conjugate) diluted 1 in 1000 in blockingbuffer is applied. Following 1 hour incubation at 37° C. the plate isdeveloped with bioFX TMB substrate. Colour development is allowed toproceed for 1-5 minutes and then stopped with stop solution. Theabsorbance is measured at 450 nm. FIG. 13 shows the VAMP cleavageactivity of LHN/B-EGF fusion protein.

Example 17 Activity of EGF-LHN/C and EGF-LHN/B in THP-1 Immune Cells

The THP-1 cell line is a human-derived suspension (non-adherent) culturethat is used frequently to provide a model system for primary monocytes.It is a well characterized model and over 2000 reviewed publicationshave utilized the THP-1 line to investigate molecular and cellularprocesses. Recent studies have demonstrated the utility of the THP-1cell line as a model to assess the secretion of anti- andpro-inflammatory cytokines (Qiu et. al. 2007 J. Lipid Res. 48(2)385-394, Prunet et. al. 2006 Cytometry A. 69, 359-373 and Segura et al2002 Clin. Exp. Immunol. 127(2) 243-254).

FIG. 14 illustrates the significant inhibition of LPS-stimulated releaseof IL-8 from THP-1 cells in culture by pretreatment with eitherEGF-LHN/C (SXN 100501) or with EGF-LHN/B (SXN 100328).

This result shows clearly the ability of fusion proteins to inhibit thepro-inflammatory cytokine secretory activity of a non-neuronal immunecell type that is a model for the monocyte cell which participates ininflammation.

Methods

THP-1, cells were pre-incubated with 10 nM compound or vehicle controlfor 48 hours at 37° C./5% CO2. After the pre-incubation, LPS was addedat a final concentration of 1 mg/ml and the cells incubated for afurther 16 hours (overnight). For inhibitory controls; cells weretreated with Staurosporine (1 μM) or Dexamethasone (1 μM) for 30 minutesprior to adding the LPS, and then incubated for 16 hours (overnight).Culture supernatant from each well was harvested and analyzed forcytokine by Luminex-based technology (BioSource). All estimations wereperformed in triplicate.

Example 18 Activity of EGF-LHN/C and EGF-LHN/B in RPMI Immune Cells

The RPMI-8226 cell line is a human-derived culture that is usedfrequently to provide a model system for primary B-lymphocytes. It is awell characterized model and over 250 reviewed publications haveutilized the RPMI-8226 line to investigate molecular and cellularprocesses. Recent studies have demonstrated the utility of the RPMI-8226cell line as a model to assess the secretion of cytokines (Xu et. al. J.Leukoc. Biol. 2002, 72(2) 410-416 and Gupta et. al. 2001, 15(12)1950-1961).

FIG. 15 illustrates the significant inhibition of LPS-stimulated releaseof IL-10 from RPMI-8226 cells in culture by pretreatment with eitherEGF-LHN/C (SXN 100501) or with EGF-LHN/B (SXN 100328).

This result shows clearly the ability of fusion proteins to inhibit thecytokine secretory activity of a non-neuronal immune cell type that is amodel for the B-lymphocyte cell which participates in immune responses.

Methods

RPMI-8226 cells were pre-incubated with 10 nM compound or vehiclecontrol for 48 hours at 37oC/5% CO2. After the pre-incubation, LPS wasadded at a final concentration of 1 mg/ml and the cells incubated for afurther 16 hours (overnight). For inhibitory controls; cells weretreated with Staurosporine (1 μM) or Dexamethasone (1 μM) for 30 minutesprior to adding the LPS, and then incubated for 16 hours (overnight).Culture supernatant from each well was harvested and analyzed forcytokine by Luminex-based technology (BioSource). All estimations wereperformed in triplicate.

Example 19 Activity of EGF-LHN/C, CP-RGD-LHN/C and EGF-LHN/B in HumanPBMC Immune Cells

PBMC are peripheral blood mononuclear cells providing a primary culturethat is highly diverse in constituent cell phenotype. It is a wellcharacterized model and over 3000 reviewed publications have utilizedprimary human PBMC to investigate molecular and cellular processes.Recent studies have demonstrated the utility of human PBMC as a model toassess the secretion of cytokines (Bachmann et. al. Cell Microbiol.2006, 8(2) 289-300, Siejka et. al. Endocr. Regul. 2005, 39(1) 7-11,Reddy et. al. 2004, 293(1-2) 127-142).

FIG. 16 illustrates the significant inhibition of LPS-stimulated releaseof IL-8 from human PBMC cells in culture by pretreatment withCP-RGD-LHN/C (SXN 100221), EGF-LHN/C (SXN 100501) or with EGF-LHN/B (SXN100328).

This result shows clearly the ability of fusion proteins to inhibit thecytokine secretory activity of non-neuronal human immune cells whichparticipates in immune responses.

Methods

PBMC cells were pre-incubated with 10 nM compound or vehicle control for24 hours at 37° C./5% CO2. After the pre-incubation, LPS was added at afinal concentration of 1 mg/ml and the cells incubated for a further 16hours (overnight). For inhibitory controls; cells were treated withStaurosporine (1 μM) or Dexamethasone (1 μM) for 30 minutes prior toadding the LPS, and then incubated for 16 hours (overnight). Culturesupernatant from each well was harvested and analyzed for cytokine byLuminex-based technology (BioSource). All estimations were performed intriplicate.

Example 20 Activity of EGF-LHN/C, CP-RGD-LHN/C and EGF-LHN/B in HumanPBMC Immune Cells

PBMC are peripheral blood mononuclear cells providing a primary culturethat is highly diverse in constituent cell phenotype. It is a wellcharacterized model and over 3000 reviewed publications have utilizedprimary human PBMC to investigate molecular and cellular processes.Recent studies have demonstrated the utility of human PBMC as a model toassess the secretion of cytokines (Bachmann et. al. Cell Microbiol.2006, 8(2) 289-300, Siejka et. al. Endocr. Regul. 2005, 39(1) 7-11,Reddy et. al. 2004, 293(1-2) 127-142).

FIG. 17 illustrates the significant inhibition of PHA-stimulated releaseof IP-10 from human PBMC cells in culture by pretreatment withCP-RGD-LHN/C (SXN 100221), EGF-LHN/C (SXN 100501) or with EGF-LHN/B (SXN100328).

This result shows clearly the ability of fusion proteins to inhibit thecytokine secretory activity of non-neuronal human immune cells whichparticipates in immune responses.

Methods

PBMC cells were pre-incubated with 10 nM compound or vehicle control for24 hours at 37° C./5% CO2. After the pre-incubation, PHA was added at afinal concentration of 2 mg/ml and the cells incubated for a further 16hours (overnight). For inhibitory controls; cells were treated withStaurosporine (1 μM) or Dexamethasone (1 μM) for 30 minutes prior toadding the PHA, and then incubated for 16 hours (overnight). Culturesupernatant from each well was harvested and analyzed for cytokine byLuminex-based technology (BioSource). All estimations were performed intriplicate.

Example 21 Clinical Example

A 54 year old male suffering from asthma presents at his GP. Despitedaily treatment with his preventer inhaler, the use of his relieverinhaler has increased significantly. The patient presents withdifficulty in performing everyday tasks due continued shortness ofbreath and frequent asthma attacks. The GP prescribes a 6-month courseof SXN100501 (as prepared in previous examples) in nebuliser form, 80 μgto be taken monthly. Following discussion with the physician, thepatient selects the most appropriate nebuliser for their personalsituation from a range of suitable devices. After a single dose ofSXN100501 the patient experiences a reduced frequency of attacks and ageneral improvement in FEV1. Further treatment enhances these parametersfurther and improves quality of life.

Example 22 Clinical Example

A 26 year old female suffering from seasonal allergic rhinitis (hayfever) presents at her GP. Despite completion of a course of preventertreatment (consisting of daily treatment with flixonase for a period of3 weeks) and subsequent treatment with OTC anti-histamines, thefrequency and severity of rhinitis increases. The GP prescribes a4-month course of SXN100328 (as prepared in previous examples), 80 μg tobe taken monthly in the form of a nasal spray. After a single dose ofSXN100328 the patient experiences a reduced frequency of rhinitis andgenerally improved quality of life. Further treatments continue todecrease the severity of the rhinitis.

SEQ ID List

-   SEQ ID NO:1 DNA sequence of LHN/B-   SEQ ID NO:2 DNA sequence of LHN/C-   SEQ ID NO:3 DNA sequence of the EGF linker-   SEQ ID NO:4 DNA sequence of the EGF-C fusion-   SEQ ID NO:5 Protein sequence of the EGF-C fusion-   SEQ ID NO:6 DNA sequence of the EGF-B fusion-   SEQ ID NO:7 Protein sequence of the EGF-B fusion-   SEQ ID NO:8 DNA sequence of the RGD linker-   SEQ ID NO:9 DNA sequence of the RGD-C fusion-   SEQ ID NO:10 Protein sequence of the RGD-C fusion-   SEQ ID NO:11 DNA sequence of the cyclic RGD linker-   SEQ ID NO:12 DNA sequence of the cyclic RGD-C fusion-   SEQ ID NO:13 Protein sequence of the cyclic RGD-C fusion-   SEQ ID NO:14 DNA sequence of the LC/C-RGD-HN/C linker-   SEQ ID NO:15 DNA sequence of the LC/C-RGD-HN/C fusion-   SEQ ID NO:16 Protein sequence of the LC/C-RGD-HN/C fusion-   SEQ ID NO:17 DNA sequence of the fully synthesised LC/C-RGD-HN/C    fusion-   SEQ ID NO:18 Protein sequence of the fully synthesised LC/C-RGD-HN/C    fusion-   SEQ ID NO:19 DNA sequence of the fully synthesised EGF-LHN/C fusion-   SEQ ID NO:20 Protein sequence of the fully synthesised EGF-LHN/C    fusion-   SEQ ID NO:21 Integrin binding peptide sequence-   SEQ ID NO:22 Integrin binding peptide sequence-   SEQ ID NO:23 Cyclic RGD peptide-   SEQ ID NO:24 Linear integrin binding sequence-   SEQ ID NO:25 Cyclic integrin binding sequence

1. A method for inhibiting secretion from a non-neuronal inflammatorycell, said method comprising administering an agent comprising at leastfirst and second domains, wherein the first domain cleaves one or moreproteins essential to exocytosis and the second domain translocates thefirst domain into the inflammatory cell.
 2. The method according toclaim 1, for treatment of disease caused, exacerbated or maintained bysecretion from said non-neuronal inflammatory cell.
 3. The methodaccording to claim 1 or 2, wherein the agent further comprises a thirddomain for targeting the agent to said non-neuronal inflammatory cell.4. The method according to claim 3 wherein the third domain comprises orconsists of a growth factor or an integrin-binding protein; or a ligandselected from (i) for mast cells, complement receptors in general,including C4 domain of the Fc IgE, and antibodies/ligands to theC3a/C4a-R complement receptor; (ii) for eosinophils, antibodies/ligandsto the C3a/C4a-R complement receptor, anti VLA-4 monoclonal antibody,anti-IL5 receptor, antigens or antibodies reactive toward CR4 complementreceptor; (iii) for macrophages and monocytes, macrophage stimulatingfactor, (iv) for macrophages, monocytes and neutrophils, bacterial IPSand yeast B-glucans which bind to CR3, (v) for neutrophils, antibody to0X42, an antigen associated with the iC3b complement receptor, or IL8;(vi) for fibroblasts, mannose 6-phosphate/insulin-like growthfactor-beta (M6P/IGFII) receptor and PA2.26, antibody to a cell-surfacereceptor for active fibroblasts in mice.
 5. The method according toclaim 1 for the treatment of a disease selected from the groupconsisting of allergies (seasonal allergic rhinitis (hay fever),allergic conjunctivitis, vasomotor rhinitis and food allergy),eosinophilia, asthma, rheumatoid arthritis, systemic lupuserythematosus, discoid lupus erythematosus, ulcerative colitis, Crohn'sdisease, hemorrhoids, pruritus, glomerulonephritis, hepatitis,pancreatitis, gastritis, vasculitis, myocarditis, psoriasis, eczema,chronic radiation-induced fibrosis, lung scarring and other fibroticdisorders.
 6. The method according to claim 1, wherein the agentcomprises a first domain that cleaves a protein selected from SNAP-25,synaptobrevin and syntaxin.
 7. The method according to claim 1 whereinthe first domain comprises a light chain of a clostridial neurotoxin, ora fragment, variant or derivative thereof which inhibits exocytosis. 8.The method according to claim 1, wherein the second domain comprises aHN region of a clostridial polypeptide, or a fragment, variant orderivative thereof that translocates the exocytosis inhibiting activityof the first domain into the cell.
 9. The method according to claim 1for inhibition of constitutive and regulated release from non-neuronalinflammatory cells.
 10. The method according to claim 1, wherein theagent is in the form of a pharmaceutical composition comprising apharmaceutically acceptable carrier.
 11. The method according to claim3, wherein the third domain is epidermal growth factor.
 12. The methodaccording to claim 3, wherein the third domain is an integrin-bindingprotein.
 13. The method according to claim 12, wherein the third domaincomprises the tri-peptide amino acid sequence Arg-Gly-Asp.
 14. Themethod according to claim 12, wherein the third domain comprises asequence selected from Arg-Gly-Asp-Phe-Val (SEQ ID NO: 23);Arg-Gly-Asp-{D-Phe}-{N-methyl-Val} (SEQ ID NO: 23); RGDFV (SEQ ID NO:23); RGDfNMeV (SEQ ID NO: 23); GGRGDMFGA (SEQ ID NO: 21); GGCRGDMFGCA(SEQ ID NO: 22); GRGDSP (SEQ ID NO: 26); GRGESP (SEQ ID NO: 27);PLAEIDGIEL (SEQ ID NO: 24) and CPLAEIDGIELC (SEQ ID NO: 25), or asequence having at least 80% identity therewith.
 15. The methodaccording to claim 2, wherein the agent further comprises a third domainfor targeting the agent to said non-neuronal inflammatory cell.